Polysaccharide compositions and methods of use for the treatment and prevention of disorders associated with progenitor cell mobilization

ABSTRACT

Polysaccharide preparations lacking substantial anticoagulant activity are provided herein. Methods of making and using such preparations are provided.

This application is a continuation-in-part of and claims priority toU.S. Ser. No. 12/762,268, filed Apr. 16, 2010, which is acontinuation-in-part of and claims priority to PCT Application No.:PCT/US2008/082223, filed Nov. 3, 2008, which claims priority to U.S.provisional application Ser. No. 60/985,123, filed Nov. 2, 2007. Thedisclosures of the prior applications are considered part of (and areincorporated by reference in) the disclosure of this application.

BACKGROUND

Heparin, a highly sulfated heparin-like glycosaminoglycan (HLGAG)produced by mast cells and isolated from natural sources, is a widelyused clinical anticoagulant. However, the effects of natural, orunfractionated, heparin can be difficult to predict and patients must bemonitored closely to prevent over- or under-anticoagulation. Lowmolecular weight heparins (LMWHs) obtained by various methods offractionation or depolymerization of polymeric heparin have morepredictable pharmacological action as anticoagulants, reduced sideeffects, sustained antithrombotic activity, and better bioavailabilitythan unfractionated heparin (UFH). Several LMWHs are approved foroutpatient treatment of thrombotic conditions.

There is increasing interest in the potential role of antithromboticagents in the management of cancer patients. Results from several recentclinical trials have suggested a survival advantage for certain types ofcancer patients treated with LMWHs (reviewed in Lemoine, 2005, Journalof Clinical Oncology, 23: 2119-20).

SUMMARY OF THE INVENTION

The invention is based, in part, on the development of polysaccharidepreparations, e.g., preparations of polysaccharides derived fromheparin, that lack substantial anticoagulant activity (e.g.,preparations of polysaccharides that have reduced anticoagulantactivity) but retain activity in other non-coagulation mediatedbiological processes. These compounds can have one or more of thefollowing features: 1) anti-Xa activity, e.g., less than 50 IU/mg, 20IU/mg, 10 IU/mg, 5 IU/mg or less, and 2) anti-metastatic,anti-angiogenic, anti-fibrotic and/or anti-inflammatory activity. Apolysaccharide preparation provided herein can also have one or more ofthe following characteristics: the preparation has glycol split uronicacid residues (e.g., less than 50%, 40%, 30%, 20% glycol split uronicacid residues); the preparation has no more than 3 glycol split uronicacid residues (U_(G)) per polysaccharide chain; the preparation hasgreater than 40% U_(2S)H_(NS,6S) disaccharide residues; degree ofdesulfation of the preparation is less than 40% (e.g., the degree ofN-desulfation is less than 40%); one or more polysaccharide chains inthe preparation have a 4,5-unsaturation of a non-reducing end uronicacid residue; one or more polysaccharide chains in the preparation havea 2,5-anhydromannitol residue at the reducing end; the weight averagemolecular weight of the preparation is between 3,500 and 8,000 Da, e.g.,between 4,000 and 8,000 Da; and a molecular weight distributiondescribed herein. This disclosure includes preparations having one ormore of these properties and characteristics as well as methods ofmaking and using such preparations.

Accordingly, in a first aspect, the invention features a polysaccharidepreparation (e.g., a heparin-derived preparation) having the followingcharacteristics: (a) a weight average molecular weight between 3,500 and8,000 Da, e.g., a weight average molecular weight described herein; (b)anti-Xa activity and/or anti-IIa activity, e.g., less than 50 IU/mg(e.g., anti-Xa activity less than about 40 IU/mg, 30 IU/mg, 20 IU/mg, 15IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg or 3 IU/mg and anti-IIa activity lessthan about 40 IU/mg, 30 IU/mg, 20 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, or3 IU/mg); and (c) less than 50% glycol split uronic acid residues (e.g.,less than 40%, 30%, 25%, or 20% glycol split uronic acid residues butmore than 1%, 5%, 10%, 15%) in the preparation. In some embodiments, thepreparation contains between 5% and 50% glycol split uronic acidresidues (e.g., between 5% and 40%, 5% and 30%, 10% and 50%, 10% and40%, 10% and 30%, or 10 and 20% glycol split uronic acid residues). Insome embodiments, the preparation has a molecular weight distributiondescribed herein.

In a second aspect, the invention features a polysaccharide preparation(e.g., a heparin-derived preparation) having the followingcharacteristics: (a) a weight average chain molecular weight between3,500 and 8,000 Da, e.g., a weight average molecular weight describedherein; (b) anti-Xa activity and/or anti-IIa activity each less than 50IU/mg (e.g., anti-Xa activity less than about 40 IU/mg, 30 IU/mg, 20IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3 IU/mg and/or anti-IIaactivity less than about 40 IU/mg, 30 IU/mg, 20 IU/mg, 10 IU/mg, 5IU/mg, 4 IU/mg, or 3 IU/mg); and (c) the polysaccharide chains of thepreparation have no more than 3 glycol split uronic acid residues(U_(G)) per polysaccharide chain (e.g., each polysaccharide chain has nomore than 2 or no more than 1 glycol split uronic acid residue (U_(G))per polysaccharide chain). The polysaccharide preparation includes oneor more chains having a glycol split uronic acid residue (U_(G)). Insome embodiments, the preparation has a molecular weight distributiondescribed herein.

In a third aspect, the invention features a polysaccharide preparation(e.g., a heparin-derived preparation) having the followingcharacteristics: (a) a weight average chain molecular weight between3,500 and 8,000 Da, a weight average molecular weight described herein;(b) anti-Xa activity and anti-IIa activity, e.g., each less than 50IU/mg (e.g., anti-Xa activity less than about 40 IU/mg, 30 IU/mg, 20IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3 IU/mg and anti-IIaactivity less than about 40 IU/mg, 30 IU/mg, 20 IU/mg, 10 IU/mg, 5IU/mg, 4 IU/mg, or 3 IU/mg); and (c) polysaccharide chains of thepreparation have on average no more than 3 glycol split uronic acidresidues (U_(G)) per polysaccharide chain (e.g., on average no more than2.5, no more than 2, no more than 1.5, or no more than 1 glycol splituronic acid residues (U_(G)) per polysaccharide chain. In someembodiments, the preparation has a molecular weight distributiondescribed herein.

In a fourth aspect, the invention features a polysaccharide preparation(e.g., a heparin-derived preparation) having the followingcharacteristics: (a) a weight average chain molecular weight between3,500 and 8,000 Da, e.g., a weight average molecular weight describedherein; (b) anti-Xa activity and anti-IIa activity, e.g., each less than50 IU/mg (e.g., anti-Xa activity less than about 40 IU/mg, 30 IU/mg, 20IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3 IU/mg and anti-IIaactivity less than about 40 IU/mg, 30 IU/mg, 20 IU/mg, 10 IU/mg, 5IU/mg, 4 IU/mg, or 3 IU/mg); and (c) the preparation has greater than40% U_(2S)H_(NS,6S) disaccharide residues (e.g., greater than 50%, 60%,70%, or 80% U_(2S)H_(NS,6S) disaccharide residues). In some embodiments,the preparation has a degree of desulfation less than 40% (e.g., lessthan 30%, 20%, or 10%). In some embodiments, the preparation has adegree of N-desulfation, 6-0 deslfation and/or 2-O desulfation of lessthan 40%, less than 30%, less than 20%, less than 10%, less than 5%,less than 2%, less than 1%. In some embodiments, the degree ofN-desulfation and 6-O desulfation is less than 40%, less than 30%, lessthan 20%, less than 10%, less than 5%, less than 2%, less than 1%. Insome embodiments, the degree of 6-O desulfation is greater than thelevel of N-desulfation. In some embodiments, the degree of N-desulfationis less then 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, the degree of 6-Odesulfation is less than 40%, 30%, 20%, 10%, 5%, and the degree of 6-Odesulfation is greater than the degree of N-desulfation. For example, inone embodiment the degree of N-desulfation is less than 5%, 4%, 3%, 2%,1% and the degree of 6-O desulfation is greater than 5%, 6%, 7%, 8%, 9%,10%, 15%. In some embodiments, the preparation has a molecular weightdistribution described herein.

In a fifth aspect, the invention features a polysaccharide preparation(e.g., a heparin-derived preparation) lacking substantial anticoagulantactivity (e.g., having reduced anticoagulant activity), wherein thepreparation includes polysaccharides that include Formula I:

[U _(w) —H _(x,y,z)]_(m) ˜[U _(G) −H _(x,y,z)]_(n)

wherein U indicates a uronic acid residue and H indicates a hexosamineresidue;

m and n are integers such that

m=4-16 (e.g., 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, or 4-15), and

n=1-4 (e.g., 1-2 or 1-3);

w=−2OS or −2OH;

x=—NS or −NAc;

y=−3OS or −3OH;

z=−6OS or −6OH;

and

wherein the symbol ˜ indicates that the units marked m and n aredistributed along the polysaccharide chain and are not necessarily insequence. For example, the following polysaccharide chain is encompassedby this embodiment:

[U _(G) −H _(x,y,z) ]−[U _(w) −H _(x,y,z) ]−[U _(G) −H _(x,y,z) ]−[U_(w) −H _(x,y,z) ]−[U _(w) −H _(x,y,z) ]−[U _(w) −H _(x,y,z)]

In addition, each of w, x, y, and z can be the same or different foreach occurrence of [U_(w)−H_(x,y,z)], and each of x, y, and z can be thesame or different for each occurrence of [U_(G)−H_(x,y,z)]. Eachoccurrence of U can independently be an iduronic acid (I) or aglucuronic acid (G). In some embodiments, the preparation has anti-Xaactivity of less than 50 IU/mg, 40 IU/mg, 30 IU/mg or 20 IU/mg butgreater than 0.1 IU/mg, 0.5 IU/mg, 1 IU/mg or 2 IU/mg and/or anti-IIaactivity of less than 50 IU/mg, 40 IU/mg, 30 IU/mg or 20 IU/mg butgreater than 0.1 IU/mg, 0.5 IU/mg, 1 IU/mg or 2 IU/mg). In someembodiments, the preparation has a molecular weight distributiondescribed herein.

In a sixth aspect, the invention features a polysaccharide preparation(e.g., a heparin-derived preparation) lacking substantial anticoagulantactivity (e.g., having substantially no anticoagulant activity) andhaving antimetastatic activity, wherein the preparation includespolysaccharides that include Formula II:

[U _(w) −H _(x,y,z)]_(m)−[U_(G) −H _(x,y,z)]_(n) −[U _(w) −H_(x,y,z)]_(o) −[U _(G) −H _(x,y,z)]_(p) −[U _(w) −H _(x,y,z)]_(q)

wherein U indicates a uronic acid residue and H indicates a hexosamineresidue;

wherein m-r are integers such that:

m=0-10;

n=0-3;

o=0-10;

p=0-3;

q=0-10;

w=−2OS or −2OH;

x=—NS or −NAc;

y=−3OS or −3OH;

z=−6OS or −6OH;

and

In some embodiments, the sum of n and p is 4, 3, 2 or 1. In someembodiments, the sum of m, o and q is between 4 and 18, e.g., 4-8, 4-9,4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16 or 4-17.

In addition, each of w, x, y, and z can be the same or different foreach occurrence of [U_(w)−H_(x,y,z)], and each of x, y, and z can be thesame or different for each occurrence of [U_(G)−H_(x,y,z)]. Eachoccurrence of U can independently be an iduronic acid (I) or aglucuronic acid (G).

In some embodiments, the preparation has anti-Xa activity of less than50 IU/mg, 40 IU/mg, 30 IU/mg or 20 IU/mg but greater than 0.5 IU/mg, 1IU/mg or 2 IU/mg and/or anti-IIa activity of less than 50 IU/mg, 40IU/mg, 30 IU/mg or 20 IU/mg but greater than 0.5 IU/mg, 1 IU/mg or 2IU/mg). In some embodiments, the preparation has a weight average chainmolecular weight between 3,500 and 8,000 Da, e.g., between 4,000 and7000 Da, 4,500 and 7,000 Da, 4,700 and 7,000 Da and 5,000 and 7,000 Da.In some embodiments, the preparation has a molecular weight distributiondescribed herein.

The invention also includes pharmaceutically acceptable salts of any ofthe preparations described herein (e.g., described above) andcompositions (e.g., pharmaceutical compositions) that comprise thepreparations described herein and/or their pharmaceutically acceptablesalts.

Any of the preparations described herein, e.g., described above, canhave other properties. E.g., one of the above described preparations orpharmaceutical compositions can further have one or more of thefunctional or structural properties set out below.

In one embodiment, at least one of the polysaccharide chains in thepreparation has one of the following structures at the non-reducing end:

wherein X is H or Me and R is H or SO₃. For example, about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or substantially all of thenon-reducing ends of the preparation or pharmaceutical composition havethe structure.

In one embodiment, at least one of the polysaccharide chains in thepreparation or pharmaceutical composition includes a 2,5-anhydromannitolresidue at the reducing end. For example, about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or substantially all of the polysaccharide chains inthe preparation or pharmaceutical composition include a2,5-anhydromannitol residue at the reducing end.

In one embodiment, the preparation or pharmaceutical composition has amolecular weight distribution such that 10-50% (e.g., 10-40%, 10-30%,15-30% or 15-25%) of the oligosaccharides of the preparation have amolecular weight<3000 Da; 40-65% (e.g., 40-60%, 45-65%, 50-65%, or55-65%) of the oligosaccharides have a molecular weight between3000-8000 Da, and 5-30% (e.g., 10-30%, 15-30%, 10-25%, or 15-25%) of theoligosaccharides have a molecular weight>8000 Da.

In one embodiment, the preparation has a polydispersity of about 1.2 to1.7 (e.g., about 1.3 to 1.7, 1.4 to 1.6, or 1.3 to 1.6).

In one embodiment, the preparation or composition has anti-metastaticactivity.

In one embodiment, the preparation or composition binds specifically toor inhibits an activity of one or more of: VEGF, FGF, SDF-1-α, HB-EGF,heparanase, SCF, sonic hedgehog, osteopontin, osteopontegerin orP-selectin.

In one embodiment, the preparation or composition has a sodium contentless than 30%, 25%, 20%, 15%, 10%. In one embodiment, the preparation orcomposition comprises: less than 20 ppm, 15 ppm, 10 ppm, 5 ppm iodine;less than 30%, 25%, 20%, 15%, 10% sulfur; less than 50, 40, 30, 20, 15ppm boron.

In one embodiment, any preparation or composition described herein ismanufactured using good manufacturing practices (GMP) as defined by theU.S. Food and Drug Administration (21 CFR Part 110).

In another aspect, the invention features methods of making apreparation. The methods include: combining UFH and nitrous acid (HONO)to produce a polysaccharide preparation; and, following nitrous acidtreatment, performing reactions to produce a glycol split of at least aportion of the uronic acid residues in the preparation.

In another aspect, methods of making a preparation include:depolymerizing an UFH (e.g., by chemical hydrolysis or enzymaticdepolymerization); and, following depolymerization, performing reactionsto produce a glycol split of at least a portion of the uronic acidresidues in the preparation.

In one embodiment, reactions to produce a glycol split of at least aportion of the uronic residues in the preparation include oxidizing thepolysaccharide preparation with periodate; and reducing the oxidizedpolysaccharide preparation with sodium borohydride. For example, themethods include oxidizing the polysaccharide preparation with periodatefor about 10-20 hours at a temperature of about 0-10° C.; and followingoxidation, reducing the sample with sodium borohydride for about 1 hourat a pH of about 5.0-8.0 at a temperature of about 0-10° C.

In another aspect, the invention features methods of manufacturing apreparation. The methods include: (1) depolymerizing an unfractionatedheparin (UFH) (e.g., by nitrous acid depolymerization, hydrolyticdepolymerization, or enzymatic depolymerization) to yield apolysaccharide preparation; (2) oxidizing the polysaccharide preparationwith periodate; (3) reducing the oxidized polysaccharide preparationwith sodium borohydride; and (4) isolating the polysaccharidepreparation (e.g., by precipitating with a salt and a polar organicsolvent, or by subjecting to a chromatographic separation orpurification), to thereby make a preparation.

In one embodiment, the step of depolymerizing includes treating the UFHwith about 0.01 to 0.05 M (e.g., about 0.02 to 0.04 M) nitrous acid at apH of about 2 to 4 for about 1 to 5 hours at a temperature of about 10to 30° C.

In one embodiment, the step of oxidizing includes treating thepolysaccharide preparation with about 0.05 to 0.2 M periodate for about10 to 20 hours at a temperature of about 0 to 10° C.

In one embodiment, the step of reducing comprises treating the oxidizedpolysaccharide preparation with about 0.5 to 2.0% (w/v) sodiumborohydride for about 0.5 to 3 hours at a pH of about 6.0 to 7.0 and atemperature of about 0 to 10° C.

In one embodiment, a method of making or manufacturing a polysaccharidepreparation includes reducing the amount of boron in the preparation.

In one embodiment, the steps in a method of manufacture described areperformed using good manufacturing practices (GMP) as defined by theU.S. Food and Drug Administration (21 CFR Part 110).

In one embodiment, the preparation is evaluated for a biologicalactivity, e.g., anti-metastatic activity; binding to any of VEGF, FGF,SDF-1α, HB-EGF, heparanase and P-selectin; or inhibition of an activityof any of VEGF, FGF, SDF-1α, and P-selectin.

The degree of desulfation, as used herein, is defined as the percentreduction in moles of sulfate per moles of disaccharide unit as comparedto unfractionated heparin.

The degree of sulfation, as used herein, is defined as the averagenumber of moles of sulfate per moles of disaccharide unit.

In another aspect, the invention features a polysaccharide preparationmade by a method described herein.

In another aspect, the invention includes an intermediate or reactionmixture from any of the methods for making or analyzing a polysaccharidepreparation described herein.

In another aspect, the invention features a pharmaceutical compositionthat includes a polysaccharide preparation described herein.

In one embodiment, the pharmaceutical composition further includes apharmaceutically acceptable carrier.

In another aspect, the invention features a method of treating a subjectthat includes administering a therapeutically effective amount of apolysaccharide preparation disclosed herein to the subject. The terms“treating”, “treatment”, and the like, mean administering thepreparation to a subject or a cell or tissue of a subject in order toobtain a desired pharmacological, physiological or clinical effect.Treatment with a polysaccharide preparation described herein may lessen,reduce, mitigate, ameliorate, delay, or prevent an existing unwantedcondition or the onset or a symptom thereof. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired pharmacological,physiological or clinical effect in the subject.

The invention includes methods for treating a subject having, or at riskof having, a metastatic disorder (e.g., a cancer, e.g., a carcinoma orother solid and hematological cancer). In those subjects, treatment mayinclude, but is not limited to, inhibited tumor growth, reduction intumor mass, reduction in size or number of metastatic lesions, inhibiteddevelopment of new metastatic lesions, prolonged survival, prolongedprogression-free survival, prolonged time to progression, and/orenhanced quality of life. In another embodiment, the subject may have adisorder or condition selected from the group consisting of: aninflammatory disorder, an autoimmune disease, a fibrotic orfibroproliferative disorder or an atopic disorder. Examples ofinflammatory disorders include but are not limited to chronicobstructive pulmonary disease, cystic fibrosis, asthma, rheumatoidarthritis, inflammatory bowel disease (including Crohns disease andulcerative colitis), multiple sclerosis, psoriasis, ischemia-reperfusioninjuries, septic shock, age-related macular degeneration (e.g., wetage-related macular degeneration), atherosclerosis, Alzheimer's disease,cardiovascular disease, vasculitis, type I and II diabetes, metabolicsyndrome, diabetic retinopathy, restenosis. Examples of autoimmunediseases include but are not limited to asthma, rheumatoid arthritis,inflammatory bowel disease, multiple sclerosis, psoriasis, type Idiabetes, systemic lupus erythematosus (SLE), Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, Guillain-Barré syndrome,autoimmune hepatitis, Myasthenia gravis. Examples of fibrotic diseasesinclude but are not limited to scleroderma, chronic obstructivepulmonary disease, diabetic nephropathy, sarcoidosis, idiopathicpulmonary fibrosis, liver fibrosis, pancreatic fibrosis, cirrhosis,cystic fibrosis, neurofibromatosis, endometriosis, post-operativefibroids, restenosis. Examples of atopic disease include but are notlimited to atopic dermatitis, atopic asthma, and allergic rhinitis. Thecompositions of the invention are administered to a subject having or atrisk of developing one or more of the diseases in an effective amountfor treating the disorder or condition.

In a preferred embodiment, the subject has, or is at risk of having, acancer or metastatic disorder (e.g., a carcinoma). For example, thesubject has a primary tumor and has, or is at risk of having, ametastasis of that primary tumor.

In one embodiment, the polysaccharide preparation is administeredintravenously or subcutaneously or is inhaled.

In one embodiment, the polysaccharide preparation is administered incombination with another therapy, e.g., another therapeutic agent, e.g.,a cytotoxic or cytostatic agent, and combinations thereof.

In one embodiment, the polysaccharide preparation is administeredchronically, e.g., at least twice over a specific period of time, e.g.,at least twice during a period of six months. In one embodiment, apolysaccharide preparation is administered twice over a period of oneweek, two weeks, three weeks, one month, two months, three months, sixmonths, one year, or even longer. The polysaccharide preparation can beadministered daily (e.g., once, twice, or three or four times daily),once every other day, weekly (e.g., once, twice, or three times a week),once every other week, once every three weeks, monthly, or any otherchronic administration schedule.

In on aspect, the invention includes methods of treating or preventing adisorder which involves or results from bone marrow derived progenitorcell mobilization. The method includes administering a polysaccharidepreparation described herein, e.g., a polysaccharide preparation thatlacks substantial anticoagulation activity, to a subject having or atrisk of having the disorder or condition.

In one embodiment, the disorder or condition involves or results frommobilization of one or more of: endothelial progenitor cells (EPCs),hematopoietic progenitor cells (HPCs), immature myeloid cells (iMC,including myeloid derived suppressor cells (MDSC) and mesenchymalprogenitor cells (MPC). In a preferred embodiment, the subject has, oris at risk of having, a cancer or metastatic disorder (e.g., acarcinoma). For example, the subject has a primary tumor and has, or isat risk of having, a metastasis of the primary tumor. In one embodiment,the subject has been or will be treated with a chemotherapeutic agentthat is associated with increased bone marrow derived progenitor cellmobilization, e.g., increased EPC, HPC, iMC and/or mesenchymalprogenitor cell mobilization. The chemotherapeutic agent can be, e.g., ataxane (e.g., paclitaxel, docetaxel, larotaxel, cabazitaxel); apyrimidine analogue (e.g., fluorouracil); an epothilone (e.g.,ixabepilone, epothilone B, epothilone D, dehydelone, sagopilone); avascular disrupting agent (e.g., AVE8062, Oxi 4503, vadimezan, ZD6126,combretastatin A-4 disodium phosphate (CA4P), DMXAA (ASA404), NPI-2358);an alkylating agent (e.g., cyclophosphamide, dacarbazine, melphalan,ifosfamide, temozolomide); an anti-angiogenic agent or a tyrosine kinaseinhibitor. In one embodiment, the anti-angiogenic agent or tyrosinekinase inhibitor selected from the group consisting of: an epidermalgrowth factor (EGF) pathway inhibitor (e.g., an epidermal growth factorreceptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF)pathway inhibitor (e.g., a vascular endothelial growth factor receptor(VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, aVEGFR-3 inhibitor)), a platelet derived growth factor (PDGF) pathwayinhibitor (e.g., a platelet derived growth factor receptor (PDGFR)inhibitor (e.g., a PDGFR-β inhibitor)), a RAF-1 inhibitor and a RETinhibitor. In some embodiments, the subject has been treated or will betreated with an anti-angiogenic agent or a tyrosine kinase inhibitorselected from the group consisting of: bevacizumab (Avastin®), imatinib(Gleevec®), cetuximab (Erbitux®), sunitinib (Sutent®), sorafenib(Nexavar®), tivozanib (AV-951), cediranib (AZD2171), dasatinib(Sprycel®), nilotinib (AMN-107), CP-547632, erlotinib (Tarceva®),panitumumab (Vectibix®), pazopanib (Votrient®), axitinib and gefitinib(Iressa®), ranibizumab (Lucentis®).

In one embodiment, the subject has been or will be treated with achemotherapeutic agent at a dose and/or dosing schedule that that isassociated with increased bone marrow derived progenitor cellmobilization. For example, the chemotherapeutic agent is a taxane (e.g.,paclitaxel, docetaxel, larotaxel, cabazitaxel) and the taxane isadministered in an amount and/or at a dosing schedule that is associatedwith increased bone marrow derived progenitor cell (e.g., EPC)mobilization, e.g., a dose and/or dosing schedule described herein. Inanother embodiment, the chemotherapeutic agent is an anti-angiogenicagent or tyrosine kinase inhibitor (e.g., an anti-angiogenic agent ortyrosine kinase inhibitor described herein, e.g., sunitinib) and theanti-angiogenic agent or tyrosine kinase inhibitor is administered in anamount and/or at a dosing schedule that is associated with increasedbone marrow derived progenitor cell (e.g., EPC) mobilization, e.g., adose and/or dosing schedule described herein. In another embodiment, thechemotherapeutic agent is a pyrimidine analogue (e.g., fluorouracil) andthe pyrimidine analogue is administered in an amount and/or at a dosingschedule that is associated with increased bone marrow derivedprogenitor cell (e.g., EPC) mobilization, e.g., a dose and/or dosingschedule described herein. In another embodiment, the chemotherapeuticagent is an anthracycline (e.g., doxorubicin) and the anthracycline isadministered in an amount and/or at a dosing schedule that is associatedwith increased bone marrow derived progenitor cell (e.g., MDSC)mobilization, e.g., a dose and/or dosing schedule described herein.

In one embodiment, the subject has cancer and has been or will beadministered a growth factor of blood cells in combination with achemotherapeutic agent. Exemplary growth factors include granulocytecolony stimulating factor (GCSF), granulocyte macrophage colonystimulating factor (GM-CSF), and erythropoietin. In one embodiment, thepolysaccharide preparation is administered after administration of thegrowth factor.

In another embodiment, the subject has cancer and has been or will beadministered a CXCR4 antagonist, e.g., in combination with achemotherapeutic agent.

In one embodiment, the cancer is a cancer described herein. For example,the cancer can be ovarian cancer, prostate cancer, lung cancer, livercancer, breast cancer, glioma, gastric cancer, pancreatic cancer, headand neck cancer, colorectal cancer, esophageal squamous cell cancer,Kaposi's sarcoma, lymphoma, multiple myeloma, melanoma, thyroidcarcinoma.

In another embodiment, the subject may have a disorder or conditionselected from the group consisting of: an inflammatory disorder, anautoimmune disease, a fibrotic or fibroproliferative disorder, avascular disorder. Examples of inflammatory disorders include, but arenot limited to, chronic obstructive pulmonary disease, asthma,rheumatoid arthritis, inflammatory bowel disease (including Crohnsdisease and ulcerative colitis), multiple sclerosis, psoriasis,ischemia-reperfusion injuries, septic shock, age-related maculardegeneration (e.g., wet age-related macular degeneration),atherosclerosis, Alzheimer's disease, cardiovascular disease,vasculitis, type I and II diabetes, metabolic syndrome, diabeticretinopathy, restenosis and eosinophilic esophagitis. Examples ofautoimmune diseases include but are not limited to asthma, rheumatoidarthritis, inflammatory bowel disease, multiple sclerosis, psoriasis,type I diabetes, systemic lupus erythematosus (SLE), Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, Guillain-Barré syndrome,autoimmune hepatitis, Myasthenia gravis. Examples of fibrotic diseasesinclude but are not limited to scleroderma, chronic obstructivepulmonary disease, diabetic nephropathy, sarcoidosis, idiopathicpulmonary fibrosis, cirrhosis, cystic fibrosis, neurofibromatosis,endometriosis, post-operative fibroids, pulmonary fibrosis, uterinefibroids, restenosis. An example of a vascular disorder is hemangioma.The compositions of the invention are administered to a subject havingor at risk of developing one or more of the disorders in an effectiveamount for treating the disorder or condition.

In another aspect, the invention features a polysaccharide preparationdescribed herein, e.g., a polysaccharide preparation the lackssubstantial anticoagulation activity described herein, for use in amethod of treatment described herein. In one embodiment, thepolysaccharide preparation can be used in any of the methods describedherein for treating or preventing a disorder which involves or resultsfrom bone marrow derived progenitor cell mobilization.

In another aspect, the invention features the use of a polysaccharidepreparation described herein, e.g., a polysaccharide preparation thatlacks substantial anticoagulation activity as described herein, formanufacture of a medicament for treating or preventing a disorderdescribed herein, e.g., for treating or preventing a disorder whichinvolves or results from bone marrow derived progenitor cellmobilization as described herein.

In another aspect, the invention features the use of a chemotherapeuticagent for the manufacture of a medicament for treating or preventing adisorder which involves or results from bone marrow derived progenitorcell mobilization as described herein, wherein the medicament is to beadministered in combination with a polysaccharide preparation describedherein, e.g., a polysaccharide preparation that lacks substantialanticoagulation activity as described herein.

In another aspect, the invention features a method of selecting apayment class for a course of treatment with a chemotherapeutic agentthat is associated with bone marrow derived progenitor cell mobilizationfor a subject, e.g., a human subject, having cancer, e.g., a cancerdescribed herein, comprising:

determining whether or not the subject is or will be receiving achemotherapeutic agent that is associated with bone marrow derivedprogenitor cell mobilization; and

assigning the subject to one of a plurality of payment classes if thesubject is receiving a chemotherapeutic agent that is associated withbone marrow derived progenitor cell mobilization wherein:

a first payment class authorizes payment for treatment of the subjectwith the chemotherapeutic agent in combination with a polysaccharidepreparation described herein, and

a second payment class authorizes payment for treatment of the subjectwith the chemotherapeutic agent without a polysaccharide preparationdescribed herein.

In some embodiments, assignment of the subject is to the first class andthe assignment authorizes payment for a course of treatment (e.g., thechemotherapeutic agent and/or a polysaccharide preparation describedherein).

In some embodiments, assignment of the subject is to the second classand the assignment authorizes a different payment for a course oftreatment (e.g., a chemotherapeutic agent in the absence of apolysaccharide preparation described herein).

In some embodiments, the method further comprises determining if thesubject has experienced a side effect from the chemotherapeutic agent.

In some embodiments, the chemotherapeutic agent can be, e.g., a taxane(e.g., paclitaxel, docetaxel, larotaxel, cabazitaxel); a pyrimidineanalogue (e.g., fluorouracil); an epothilone (e.g., ixabepilone,epothilone B, epothilone D, dehydelone, sagopilone); a vasculardisrupting agent (e.g., AVE8062, Oxi 4503, vadimezan, ZD6126,combretastatin A-4 disodium phosphate (CA4P), DMXAA (ASA404), NPI-2358);an alkylating agent (e.g., cyclophosphamide, dacarbazine, melphalan,ifosfamide, temozolomide); an anti-angiogenic agent or a tyrosine kinaseinhibitor, e.g., an anti-angiogenic agent or tyrosine kinase inhibitordescribed herein.

For any of the ranges described herein, e.g., for a given structure oractivity, the ranges can be those ranges disclosed as well as otherranges. For example, a range constructed from a lower endpoint of onerange, e.g., for a given building block or activity, can be combinedwith the upper endpoint of another range, e.g., for the given buildingblock or activity, to give a range.

An “isolated” or “purified” polysaccharide preparation is substantiallyfree of cellular material or other contaminating proteins from the cellor tissue source from which the polysaccharide is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. “Substantially free” means that a preparation isat least 50% pure (wt/wt). In a preferred embodiment, the preparationhas less than about 30%, 20%, 10% and more preferably 5% (by dryweight), of non-heparin-derived polysaccharides, proteins or chemicalprecursors or other chemicals, e.g., from manufacture. These are alsoreferred to herein as “contaminants.” Examples of contaminants that canbe present in a polysaccharide preparation provided herein include, butare not limited to, sodium, sulfur, boron, enzyme (e.g., a heparinaseenzyme), methanol, ethanol, iodine, and chloride.

“Combined use” or “in combination” as used herein means that theindividual agents are administered concurrently or within a timeinterval such that the use of the combined agents provides an increasedbenefit (e.g., increased efficacy or decreased side effects) than ifthey were administered otherwise. In one embodiment, the individualagents are administered within an interval such that the physiologicaleffects of the agents on the subject overlap.

The term “payment class,” as used herein, refers to payment plancorrelated with a treatment regimen. The payment plan can be, e.g.,payment for a treatment, a level of payment for a treatment,reimbursement for a treatment, a level of reimbursement for a treatment,denial of a payment for a treatment, denial of reimbursement for atreatment or denial of coverage for a treatment.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of a polysaccharide preparationdescribed herein in a murine melanoma experimental metastasis (B16F101.v.) model. Lung tumor burden (lung weight-normal lung weight) wasdetermined for female C57BL/6 mice (9-10 weeks old) challenged with i.v.injection of 2×10⁵ B16F10 cells and pretreated with a single dose (10mg/kg) of MONC402 (batch R-1-5), dalteparin (Fragmin®), or MONC 202(negative control, N-desulfated polysaccharide) immediately beforeinjection. “Normal” designates unchallenged and untreated mice.

FIG. 2 is a bar graph showing the effect of a polysaccharide preparationdescribed herein in a 4T1 therapeutic model of breast cancer metastasisto the lung. Lung tumor burden (lung weight-normal lung weight) wasdetermined on day 32 for female BALB/c mice (8 weeks old) challengedwith intra-mammary fat pad injection of 8×10⁴ 4T1 cells and treated asindicated starting on day 4.

FIG. 3 depicts the effect of MONC402 on G-CSF or docetaxel induced EPCmobilization.

FIG. 4 depicts the effect of MONC402 on G-CSF or docetaxel induced EPCmobilization.

FIG. 5 depicts the effect of MONC402 on docetaxel induced EPCmobilization in tumor-bearing mice.

FIG. 6 depicts the effect of G-CSF on MSDC mobilization.

FIG. 7 depicts effect of sunitinib (Sutent®) on EPC mobilization.

FIG. 8 is a graph depicting the effect of MONC402 on sunitinib(Sutent®)-induced accelerated tumor regrowth and metastasis.

DETAILED DESCRIPTION Polyanions/Polysaccharides

The methods described herein relate to combination therapies including apolyanion such as a polysaccharide, glycosaminoglycan (GAGs), heparin,low molecular weight heparin, chemically or enzymatically modifiedheparin or heparin sulfate, heparan sulfate mimetic (e.g., PI-88),chemically or enzymatically synthesized polysaccharide, e.g., K5polysaccharide. In one embodiment, the polyanion, polysaccharide, GAG,heparin, low molecular weight heparin, chemically or enzymaticallymodified heparin or heparan sulfate, heparan sulfate mimetic orchemically or enzymatically synthesized polysaccharide lacks substantialanticoagulant activity, i.e., exhibits less than 50 IU/mg of anti-IIaactivity and less than 50 IU/mg of anti-Xa activity. In one embodiment,the polyanion, polysaccharide, GAG, heparin, low molecular weightheparin, chemically or enzymatically modified heparin or heparansulfate, heparan sulfate mimetic or chemically or enzymaticallysynthesized polysaccharide exhibits residual anticoagulant activity,e.g., exhibits at least 0.1 IU/mg anti-IIa activity and at least 0.1IU/mg anti-Xa activity, or at least 0.2 IU/mg anti-IIa activity and atleast 0.2 IU/mg anti-Xa activity, or at least 0.5 IU/mg anti-IIaactivity and at least 0.5 IU/mg anti-Xa activity, or at least 1 IU/mganti-IIa activity and at least 1 IU/mg anti-Xa activity. In someembodiments, the polyanion, polysaccharide, GAG, heparin, low molecularweight heparin, chemically or enzymatically modified heparin or heparansulfate, heparan sulfate mimetic or chemically or enzymaticallysynthesized polysaccharide exhibits 2 IU/mg, 3 IU/mg, 4 IU/mg, 5 IU/mg,6 IU/mg, 7 IU/mg, 8 IU/mg, 9 IU/mg, 10 IU/mg, 12 IU/mg, 15 IU/mg, 18IU/mg, 20 IU/mg, 22 IU/mg, 25 IU/mg, 28 IU/mg, 30 IU/mg of anti-IIaactivity. In some embodiments, the polyanion, polysaccharide, GAG,heparin, low molecular weight heparin, chemically or enzymaticallymodified heparin or heparan sulfate, heparan sulfate mimetic orchemically or enzymatically synthesized polysaccharide exhibits 2 IU/mg,3 IU/mg, 4 IU/mg, 5 IU/mg, 6 IU/mg, 7 IU/mg, 8 IU/mg, 9 IU/mg, 10 IU/mg,12 IU/mg, 15 IU/mg, 18 IU/mg, 20 IU/mg, 22 IU/mg, 25 IU/mg, 28 IU/mg, 30IU/mg of anti-Xa activity.

Heparin Preparations

In some aspects, the methods and kits described herein include a heparinpreparation. A heparin preparation, as used herein, is a preparationwhich contains heparin or a preparation derived therefrom. Heparinpreparations include unfractionated heparin preparations, low molecularweight heparin (LMWH) preparations, ultra low molecular weight heparin(ULMWH) preparations and the like.

The term “unfractionated heparin (UFH)” as used herein, is heparinpurified from porcine intestinal mucosa. UFH can be used, e.g., as astarting material in the process to form a LMWH or an ULMWH. UFH iscommercially available from several vendors including Abbott, Organon,Riker, Invenex, Baxter, Calbiochem, Sigma or Upjohn.

Examples of LMWH preparations include, but are not limited to, anenoxaparin preparation (Lovenox™ or Clexane™); a dalteparin preparation(Fragmin™); a certoparin preparation (Sandoparin™ or Embollex); anardeparin preparation (Normiflo™); a nadroparin preparation(Fraxiparin™); a parnaparin preparation (Fluxum™); a reviparinpreparation (Clivarin™); a tinzaparin preparation (Innohep™ orLogiparin™), a fondaparinux preparation (Arixtra™), or a M118-REHpreparation. In some embodiments, the LMWH is a LMWH other than anenoxaparin preparation (Lovenox™ or Clexane™); a dalteparin preparation(Fragmin™); a certoparin preparation (Sandoparin™ or Embollex); anardeparin preparation (Normiflo™); a nadroparin preparation(Fraxiparin™); a parnaparin preparation (Fluxum™); a reviparinpreparation (Clivarin™); a tinzaparin preparation (Innohep™ orLogiparin™), a fondaparinux preparation (Arixtra™), or a M118-REHpreparation.

Polysaccharide Preparations that Lack Substantial AnticoagulationActivity

In many clinical settings, commercially available LMWH preparations arepreferred over UFH preparations as anticoagulants because LMWHs havemore predictable pharmacokinetics and can be administeredsubcutaneously. However, because of the potential for bleedingcomplications due to their anticoagulant effects, currently availableLMWH preparations are less suitable for therapy of non-coagulationmediated disorders, and/or for disorders that may require higher dosesor chronic dosing regimens. The invention features polysaccharidepreparations designed to lack substantial anticoagulant activity whileretaining clinically advantageous properties. Properties of thepolysaccharide preparations include, e.g., lacking substantialanticoagulant activity, e.g., anti-IIa activity less than 50 IU/mg,anti-Xa activity less than 50 IU/mg), and having anti-metastatic,anti-angiogenic, anti-fibrotic and/or anti-inflammatory activity.

Examples of such polysaccharide preparations include chains that includethe following:

[U _(w) −H _(x,y,z)]_(m) ˜[U _(G) −H _(x,y,z)]_(n)

wherein U indicates a uronic acid residue and H indicates a hexosamineresidue, wherein m and n are integers such that m=6-18, and n=1-4,w=−2OS or −2OH, x=—NS or —NAc, y=−3OS or −3OH, z=−6OS or −6OH,

and

wherein the symbol ˜ indicates that the units marked m and n aredistributed along the polysaccharide chain and are not necessarily insequence. For example, the following polysaccharide chain is encompassedby this embodiment:

[U _(G) −H _(x,y,z) ]−[U _(w) −H _(x,y,z) ]−[U _(G) −H _(x,y,z) ]−[U_(w) −H _(x,y,z) ]−[U _(w) −H _(x,y,z) ]−[U _(w) −H _(x,y,z)]

In addition, each of w, x, y, and z can be the same or different foreach occurrence of [U_(w)H_(x,y,z)], and each of x, y, and z can be thesame or different for each occurrence of [U_(G)−H_(x,y,z)]. Eachoccurrence of U can independently be an iduronic acid (I) or aglucuronic acid (G).

The polysaccharide preparation can have anti-Xa activity and anti-IIaactivity each less than 50 IU/mg (e.g., anti-Xa activity less than about40 IU/mg, 30 IU/mg, 20 IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3IU/mg, 2 IU/mg or 1 IU/mg; or from about 0 to 50 IU/mg, about 0 to 40IU/mg, about 0 to 30 IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg,about 0 to 10 IU/mg, about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to15 IU/mg, or about 5 to 20 IU/mg; and anti-IIa activity less than about40 IU/mg, 30 IU/mg, 20 IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3IU/mg, 2 IU/mg or 1 IU/mg; or from about 0 to 50 IU/mg, about 0 to 40IU/mg, about 0 to 30 IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg,about 0 to 10 IU/mg, about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to15 IU/mg, or about 5 to 20 IU/mg); and

[U _(w) −H _(x,y,z)]_(m) −[U _(G) −H _(x,y,z)]_(n) −[U _(w) −H_(x,y,z)]_(o) −[U _(G) −H _(x,y,z)]_(p) −[U _(w) −H _(x,y,z)]_(q)

wherein U indicates a uronic acid residue and H indicates a hexosamineresidue, wherein m-r are integers such that: m=0-10, n=0-3, o=0-10,p=0-3, q=0-10, w=−2OS or −2OH, x=—NS or —NAc, y=−3OS or −3OH, z=−6OS or−6OH,

and

wherein w, x, y, and z are each the same or different on each unitmarked m, n, o, p, or q. In some embodiments, the sum of n+p is lessthan or equal to 4 (e.g., less than or equal to 3, 2, 1, or 0). In someembodiments, the sum of n and p is 4, 3, 2 or 1. In some embodiments,the sum of m, o and q is between 4 and 18, e.g., 4-8, 4-9, 4-10, 4-11,4-12, 4-13, 4-14, 4-15, 4-16 or 4-17.

In addition, each of w, x, y, and z can be the same or different foreach occurrence of [U_(w)−H_(x,y,z)], and each of x, y, and z can be thesame or different for each occurrence of [U_(G)−H_(x,y,z)]. Eachoccurrence of U can independently be an iduronic acid (I) or aglucuronic acid (G).

The polysaccharide preparation can have anti-Xa activity and anti-IIaactivity each less than 50 IU/mg (e.g., anti-Xa activity less than about40 IU/mg, 30 IU/mg, 20 IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3IU/mg, 2 IU/mg or 1 IU/mg; or from about 0 to 50 IU/mg, about 0 to 40IU/mg, about 0 to 30 IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg,about 0 to 10 IU/mg, about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to15 IU/mg, or about 5 to 20 IU/mg; and anti-IIa activity less than about40 IU/mg, 30 IU/mg, 20 IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3IU/mg, 2 IU/mg or 1 IU/mg; or from about 0 to 50 IU/mg, about 0 to 40IU/mg, about 0 to 30 IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg,about 0 to 10 IU/mg, about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to15 IU/mg, or about 5 to 20 IU/mg). In some embodiments, the preparationhas a weight average chain molecular weight between 3,500 and 7,000 Da,e.g., 4,300 and 7000 Da, 4,500 and 7,000 Da, 4,700 and 7,000 Da and5,000 and 7,000 Da.

Anti-IIa Activity

Polysaccharide preparations are disclosed herein that providesubstantially reduced anti-IIa activity, e.g., e.g., anti-IIa activityof about less than about 50 IU/mg, less than about 40 IU/mg, 30 IU/mg,20 IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3 IU/mg, 2 IU/mg or 1IU/mg; or from about 0 to 50 IU/mg, about 0 to 40 IU/mg, about 0 to 30IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg, about 0 to 10 IU/mg,about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to 15 IU/mg, or about 5to 20 IU/mg). Anti-IIa activity is calculated in International Units ofanti-IIa activity per milligram using statistical methods for parallelline assays. The anti-IIa activity levels described herein are measuredusing the following principle.

IIa (Excess)+Substrate Peptide+pNA (measured spectrophotometrically)Anti-factor IIa activity is determined by the sample potentiating effecton antithrombin (ATIII) in the inhibition of thrombin. Thrombin excesscan be indirectly spectrophotometrically measured. The anti-factor IIaactivity can be measured, e.g., on a Diagnostica Stago analyzer or on anACL Futura3 Coagulation system, with reagents from Chromogenix (S-2238substrate, Thrombin (53 nkat/vial), and Antithrombin), or on anyequivalent system. Analyzer response is calibrated using the 2ndInternational Standard for Low Molecular Weight Heparin.

Anti-Xa Activity

Preferably, a polysaccharide preparation provided herein has anti-Xaactivity of about 0 to 50 IU/mg, e.g., 50 IU/mg, 40 IU/mg, 30 IU/mg, 20IU/mg, 15 IU/mg, 10 IU/mg, 5 IU/mg, 4 IU/mg, 3 IU/mg, 2 IU/mg or 1IU/mg; or from about 0 to 50 IU/mg, about 0 to 40 IU/mg, about 0 to 30IU/mg, about 0 to 25 IU/mg, about 0 to 20 IU/mg, about 0 to 10 IU/mg,about 0 to 5 IU/mg, about 5 to 10 IU/mg, about 5 to 15 IU/mg, or about 5to 20 IU/mg). Anti-Xa activity of a preparation is calculated inInternational Units of anti-factor Xa activity per milligram usingstatistical methods for parallel line assays. The anti-factor Xaactivity of preparations described herein is measured using thefollowing principle:

FXa (Excess)+Substrate→Peptide+pNA (measured spectrophotometrically)

The anti-factor Xa activity is determined by the sample potentiatingeffect on antithrombin (ATIII) in the inhibition of activated Factor Xa(FXa). Factor Xa excess can be indirectly spectrophotometricallymeasured. Anti-factor Xa activity can be measured, e.g., on aDiagnostica Stago analyzer with the Stachrom® Heparin Test kit, on anACL Futura3 Coagulation system with the Coatest® Heparin Kit fromChromogenix, or on any equivalent system. Analyzer response can becalibrated using the NIBSC International Standard for Low MolecularWeight Heparin.

Molecular Weight and Chain Length

When weight average molecular weight of a preparation is determined, aweight average molecular weight of about 3500 to 8000 Da, about 3500 to7000 Da, preferably about 4000 to 7000 Da, about 4200 to 6000, or about4500 to 6000 Da, indicates that a significant number of chains in thepolysaccharide preparation are of sufficient chain length.

“Weight average molecular weight” as used herein refers to the weightaverage in daltons of chains of uronic acid/hexosamine disacchariderepeats. The presence of non-uronic acid and/or non-hexosamine buildingblocks are not included in determining the weight average molecularweight. Thus, the molecular weight of non-uronic acid and non-hexosaminebuilding blocks within a chain or chains in the preparation should notbe included in determining the weight average molecular weight. Theweight average molecular weight (M_(w)) is calculated from the followingequation: M_(w)=Σ(c_(i)m_(i))/Σc_(i). The variable c_(i) is theconcentration of the polymer in slice i and m_(i) is the molecularweight of the polymer in slice i. The summations are taken over achromatographic peak, which contains many slices of data. A slice ofdata can be pictured as a vertical line on a plot of chromatographicpeak versus time. The elution peak can therefore be divided into manyslices. The weight average molecular weight calculation is averagedependant on the summation of all slices of the concentration andmolecular weight. The weight average molar weight can be measured, e.g.,using the Wyatt Astra software or any appropriate software. The weightaverage molecular weights described herein are determined by high liquidchromatography with two columns in series, for example a TSK G3000 SWXLand a G2000 SWXL, coupled with a UV or multi angle light scattering(MALS) detector and a refractometric detector in series. The eluent usedis a 0.2 M sodium sulfate, pH 5.0, and a flow rate of 0.5 mL/min.

A determination of whether a polysaccharide preparation includes chainsof sufficient chain length can be made, for example, by determining theaverage chain length of the chains in the preparation and/or bydetermining the weight average molecular weight of chains within thepreparation. When average chain length is determined, an average chainlength of about 5 to 22, e.g., about 7 to 18, typically about 7 to 14 or8 to 13 disaccharide repeats, indicates that a significant number ofchains in the preparation are of sufficient chain length.

“Average chain length” as used herein refers to the average chain lengthof uronic acid/hexosamine disaccharide repeats that occur within achain. The presence of non-uronic acid and/or non-hexosamine buildingblocks (e.g., attached PEG moieties) are not included in determining theaverage chain length. Average chain length is determined by dividing thenumber average molecular weight (Mn) by the number average molecularweight for a disaccharide (500 Da).

Glycol Split Uronic Acids

A polysaccharide preparation described herein can include an opening ofthe glycoside ring, conventionally called reduction-oxidation (RO)derivatives. In these preparations, one or more glycoside rings havingvicinyl diols that are opened, e.g., at the bond between C2 and C3, bymeans of an oxidation action, followed by a reduction. The compoundsreferred to herein will also be called “Glycol Split” derivatives.

In a further embodiment of the invention described herein, the glycolsplit residues lend themselves to the subsequent functionalization.Therefore, the compounds may also bear equal or different groups, inplace of the primary hydroxy groups deriving from glycol split, forexample, aldehyde groups, methoxy groups, or oligosaccharide or peptidegroups, ranging from a single saccharide or amino acid to more than oneunit of length, e.g., 2 or 3 units.

In some embodiments, fewer than 50% of the uronic acid residues areglycol split uronic acid residues (e.g., less than 40%, 30%, 25%, or 20%of the uronic acid residues are glycol split uronic acid residues).

Reducing End Structures

In some instances, at least about 50% of the chains in a polysaccharidepreparation described herein have a modified reducing end structure suchas a 2,5-anhydromannose residue or a 2,5-anhydromannose that has beenreduced to form an alcohol. In some embodiments, at least about 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the chains in thepreparation have a modified reducing end structure, such that thereducing end includes a 2,5-anhydromannose residue or a2,5-anhydromannose that has been reduced to form an alcohol.

Polydispersity

The polydispersity of polysaccharide preparations provided herein isabout 2 or less, e.g., 1.7 or less, e.g., about 1.7 or 1.6 to 1.2, about1.4-1.5, and numbers in between.

The term “polydisperse” or “polydispersity” refers to the weight averagemolecular weight of a composition (Mw) divided by the number averagemolecular weight (Mn). The number average molecular weight (Mn) iscalculated from the following equation: Mn=Σci/(Σci/mi). The variable ciis the concentration of the polysaccharide in slice i and Mi is themolecular weight of the polysaccharide in slice i. The summations aretaken over a chromatographic peak, which contains many slices of data. Aslice of data can be pictured as a vertical line on a plot ofchromatographic peak versus time. The elution peak can therefore bedivided into many slices. The number average molecular weight is acalculation dependent on the molecular weight and concentration at eachslice of data. Methods of determining weight average molecular weightare described above, and were used to determine polydispersity as well.

Methods of Making Polysaccharide Preparations

Various methods of making polysaccharide preparations, e.g., apreparation described herein, are also contemplated. One method includesproviding a precursor heparin preparation having a weight averagemolecular weight of greater than 7000 Da or a chain length of greaterthan 7 to 18 disaccharides, and processing the precursor heparinpreparation (e.g., by enzymatic or chemical depolymerization, e.g., bynitrous acid depolymerization) to obtain a polysaccharide preparationhaving a weight average molecular weight of about 3000 to 8000 Da or anaverage chain length of about 7 to 18 disaccharides. For example, theprecursor heparin preparation can be unfractionated heparin.

The precursor heparin preparation can be processed by a methodcomprising depolymerization (e.g., by nitrous acid treatment,hydrolysis, or enzymatic depolymerization) followed by a glycol splitreaction. Nitrous acid depolymerization can be accomplished, e.g., bytreating the precursor heparin preparation (e.g., UFH) with nitrous acid(e.g., about 0.02 to 0.04 M nitrous acid) at a pH of about 2 to 4 for aspecified period of time (e.g., about 1 to 5 hours) at a temperature ofabout 10 to 30° C. The glycol split reaction involves periodateoxidation using periodate (e.g., about 0.05 M to 0.2 M sodium periodate)for about 10 to 20 hours at a temperature of about 0 to 10° C. In someembodiments, residual impurities such as salts or diethylene glycol(DEG) can be subsequently removed by a chromatographic method, e.g. gelfiltration chromatography. Optionally, the oxidized preparation is thenreduced by treatment with a reducing agent (e.g., about 0.5 to 2.0%(w/v) sodium borohydride) for about 0.5 to 3 hours at a pH of about 6.0to 7.0 and a temperature of about 0 to 10° C.

A precursor heparin preparation can be processed using enzymaticdigestion, chemical digestion or combinations thereof. Examples ofchemical digestion include oxidative depolymerization, e.g., with H₂O₂or Cu⁺ and H₂O₂, deaminative cleavage, e.g., with isoamyl nitrite ornitrous acid, β-eliminative cleavage, e.g., with benzyl ester, and/or byalkaline treatment. Enzymatic digestion can include the use of one ormore heparin degrading enzymes. For example, the heparin degradingenzyme(s) can be, e.g., one or more heparanase, heparin lyase, heparansulfate glycosaminoglycan (HSGAG) lyase, a lyase described as aglycosaminoglycan (GAG) lyase that can also degrade heparin. Preferably,the enzyme cleaves at one or more glycosidic linkages of unsulfateduronic acids.

Biological Activities

The preparations described herein have anti-metastatic activity asassayed in an animal model of metastasis in which B16F10 melanoma cellsinjected into the tail veins of C57BL/6 mice arrest in the lungs andproliferate as discrete pulmonary foci. This assay is generallydescribed in Gabri et al., 2006, Clin. Cancer Res., 12:7092-98. Apreparation may additionally have activity in other experimental modelsof metastasis, including the C170HM2 assay, in which C170HM2 humancolorectal cancer line cells are injected into the peritoneal cavity,where the primary site of metastasis is to the liver. The preparationsdescribed herein may also show anti-metastatic activity in spontaneousmodels of metastasis, such as the APSLV model, in which APSLV humancolorectal cancer cells are implanted into the peritoneal wall andexhibit spontaneous metastasis to the lung, or the 4T1 model, in which4T1 murine mammary carcinoma cells implanted in to the mammary fat padexhibit spontaneous metastasis to the lung and other organs.

The preparations described herein can bind to and/or modulate (e.g.,inhibit) an activity of one or more of VEGF, FGF, SDF-1α, HB-EGF,heparanase and P-selectin. In some embodiments, interaction of thepreparation with (e.g., binding to) a target protein (e.g., VEGF, FGF,SDF-1α, or P-selectin) can be assayed, e.g., in vitro, e.g., usingmethods known in the art. Numerous methods and techniques to detectbinding or modulation (e.g., inhibition) of activity are known, e.g.,standard receptor competition assays, fluorescence energy transfer(FET), fluorescence resonance energy transfer (FRET) (see, for example,U.S. Pat. No. 5,631,169; U.S. Pat. No. 4,868,103), and fluorescencepolarization (FP). In some embodiments, evaluating binding of apolysaccharide preparation to a target protein can include a real-timemonitoring of the binding interaction, e.g., using BiomolecularInteraction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky (1991)Anal. Chem., 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol., 5:699-705). Surface plasmon resonance or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore).

Activities of VEGF, FGF, and P-selectin on cells in vitro and in vivoare well known in the art. The ability of a polysaccharide preparationto modulate (e.g., inhibit) an activity of VEGF, FGF, or P-selectin canbe assayed in vitro or in a cell-based assay or in vivo in an organism.For example, the ability of a polysaccharide preparation to modulate(e.g., inhibit) the activity of VEGF, FGF, or P-selectin to modulate(e.g., stimulate) the proliferation of endothelial cells, e.g., humanumbilical vein epithelial cells, can be assayed. Exemplary methods ofdetermining modulation of FGF activity can be found in U.S. Pat. No.5,733,893. A cell-based assay can be performed using a single cell, or acollection of at least two or more cells. The cell can be a yeast cell(e.g., Saccharomyces cerevisiae) or a mammalian cell, e.g., a cell line.

Assays for determining whether a chemotherapeutic agent causes bonemarrow derived progenitor cell mobilization can be determined by methodsknown in the art, see, e.g., Shaked et al. (2008) Cancer Cell14:263-273, which is incorporated herein by reference, and described inthe Examples.

Pharmaceutical Compositions

Compositions, e.g., pharmaceutically acceptable compositions, whichinclude a preparation described herein, formulated together with apharmaceutically acceptable carrier, are provided.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, isotonic and absorption delaying agents,and the like that are physiologically compatible with parenteraladministration. The carrier can be suitable for any parenteraladministration, e.g., intravenous, intramuscular, subcutaneous,intraocular, intraperitoneal, rectal, inhaled or spinal administration(e.g., by injection or infusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, and liposomes. The preferred form depends on theintended mode of administration and therapeutic application. Typicalpreferred compositions are in the form of injectable or infusiblesolutions. The preferred mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular).In a preferred embodiment, the preparation is administered byintravenous infusion or injection. In another preferred embodiment, thepreparation is administered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, subcutaneous, intraarterial,intrathecal, intracapsular, intraorbital, intravitreous, intracardiac,intradermal, intraperitoneal, transtracheal, inhaled, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,polysaccharide preparation) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying that yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example, variouspolymers, monostearate salts and gelatin.

For many therapeutic applications, the preferred route/mode ofadministration is intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results.

Formulations for injection may be presented in unit dosage form, e.g.,in ampoules, syringes, syringe pens, or in multi-dose containers, e.g.,with an added preservative. The compositions may take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and maycontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

For administration by inhalation, the preparation may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. In addition, dry powder formations for inhalationtherapy are within the scope of the invention. Such dry powderformulations may be prepared as disclosed, e.g., in WO 02/32406.

In addition to the compositions described previously, the compounds mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. The compositions can be included in acontainer, pack, or dispenser together with instructions foradministration.

The preparation can also be administered with short or long termimplantation devices, e.g., a stent. The preparation can be implantedsubcutaneously, can be implanted into tissues or organs (e.g., thecoronary artery, carotid artery, renal artery and other peripheralarteries, veins, kidney, heart cornea, vitreous, cerebrum, etc.), or canbe implanted in physiological spaces around tissues and organs (e.g.,kidney capsule, pericardium, thoracic or peritoneal space).

The preparation can also be used to coat various medical devices. Forexample, the preparation can be used to coat a stent or extracorporealcircuit. Such formulations of the preparations may include using, e.g.,controlled release beads, gel or microspheres as well as variouspolymers such as PLGA, cellulose, alginate or other polysaccharides.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions.

The pharmaceutical compositions of the invention may include atherapeutically effective amount of a preparation. A therapeuticallyeffective amount of the preparation may vary according to factors suchas the disease state, age, sex, and weight of the individual and caninclude more than one unit dose. A therapeutically effective amount isalso one in which any toxic or detrimental effects of the preparationare outweighed by the therapeutically beneficial effects. Atherapeutically effective amount may inhibit a measurable parameter,e.g., VEGF activity, FGF activity, P-selectin activity, heparanaseactivity, or size or rate of growth of metastatic lesions, e.g., by atleast about 20%, more preferably by at least about 25%, 30%, 40%, evenmore preferably by at least about 50%, 60%, and still more preferably byat least about 70%, 80% relative to untreated subjects. The ability of acompound to inhibit a measurable parameter, e.g., metastasis orangiogenesis, can be evaluated in an animal model system or in a human(e.g., in a pre-clinical model or a clinical trial). Alternatively, aproperty of a composition can be evaluated by examining the activity ofthe compound in an in vitro assay. Exemplary doses for intravenous orsubcutaneous administration of the polysaccharide preparation are about0.03 mg/kg to 0.45 mg/kg, e.g., 0.03 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15mg/kg, 0.2 mg/kg, 0.22 mg/kg, 0.25 mg/kg, 0.27 mg/kg, 0.3 mg/kg, 0.35mg/kg, 0.37 mg/kg, 0.4 mg/kg, 0.44 mg/kg, preferably about 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.44 mg/kg, 0.47 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.7 mg/kg,preferably about 0.30 to 0.50 mg/kg, e.g., 0.30 mg/kg, 0.35 mg/kg, 0.40mg/kg, 0.42 mg/kg, 0.44 mg/kg, 0.47 mg/kg or 0.50 mg/kg. In someembodiments, the polysaccharide preparation can be administered at adose between 0.5-80 mg/kg, between 0.5-40 mg/kg, between 0.5-30 mg/kg,e.g., between 5-50 mg/kg/day.

Kits

Also within the scope of the invention are a kit comprising apolysaccharide preparation described herein, e.g., a polysaccharidepreparation described herein that lacks substantial anticoagulationactivity, and a chemotherapeutic agent that is associated with bonemarrow derived progenitor cell mobilization; a kit comprising apolysaccharide preparation described herein, e.g., a polysaccharidepreparation that lacks substantial anticoagulation activity, andinstructions to administer the polysaccharide preparation to a subjectwith cancer who has been or will be treated with a chemotherapeuticagent that is associated with bone marrow derived progenitor cellmobilization; a kit comprising a polysaccharide preparation describedherein, e.g., a polysaccharide preparation that lacks substantialanticoagulation activity, and instructions to administer thepolysaccharide preparation to a subject with cancer who has been or willbe treated with a chemotherapeutic agent at a dose and/or dosingschedule that is associated with bone marrow derived progenitor cellmobilization, e.g., a dose and/or dosing schedule described herein; akit comprising a chemotherapeutic agent that is associated with bonemarrow derived progenitor cell mobilization, and instructions toadminister the chemotherapeutic agent to a subject in combination with apolysaccharide preparation described herein, e.g., a polysaccharidepreparation described herein that lacks substantial anticoagulantactivity; or a kit comprising a chemotherapeutic agent, and instructionsto administer the chemotherapeutic agent to a subject at a dose and/ordosing schedule associated that is associated with bone marrowprogenitor cell mobilization and instructions to administer thechemotherapeutic agent in combination with a polysaccharide preparationdescribed herein, e.g., a polysaccharide preparation described hereinthat lacks substantial anticoagulant activity.

The kit can include one or more other elements including: otherreagents, e.g., a therapeutic agent; devices or other materials forpreparing the polysaccharide preparation for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject. The instructions can include instructionsfor therapeutic application including suggested dosages and/or modes ofadministration, e.g., in a patient having a disorder, e.g., a disorderdescribed herein. The kit can further contain at least one additionalreagent, such as a diagnostic or therapeutic agent, e.g., a diagnosticor therapeutic agent as described herein, formulated as appropriate, inone or more separate pharmaceutical preparations.

Uses

The polysaccharide preparations can be used to treat a subject. As usedherein, a subject is a mammal, e.g., a non-human experimental mammal, aveterinary mammal, or a human. Non-human mammals include a primate, cow,horse, pig, sheep, goat, dog, cat, or rodent.

The preparations provided herein can be used, for example, to treat orprevent a metastatic disorder (e.g., a cancer, e.g., a carcinoma orother solid or hematological cancer). As used herein, the term “cancer”is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. Methods and compositions disclosed herein are particularlyuseful for treating, or reducing the size, numbers, or rate of growthof, metastatic lesions associated with cancer.

Examples of cancers include, but are not limited to, solid tumors, softtissue tumors, hematopoietic tumors and metastatic lesions. Examples ofsolid tumors include malignancies, e.g., sarcomas, adenocarcinomas, andcarcinomas, of the various organ systems, such as those affecting headand neck (including pharynx), thyroid, lung (small cell or non smallcell lung carcinoma), breast, lymphoid, gastrointestinal (e.g., oral,esophageal, stomach, liver, pancreas, small intestine, colon and rectum,anal canal), genitals and genitourinary tract (e.g., renal, urothelial,bladder, ovarian, uterine, cervical, endometrial, prostate, testicular),CNS (e.g., neural or glial cells, e.g., neorublastoma or glioma), skin(e.g., melanoma). Examples of hematopoietic cancers that can be treatedinclude hemangiomas, multiple myeloma, lymphomas and leukemias andmyelodysplasia. Methods and compositions disclosed herein areparticularly useful for treating, e.g., reducing or delaying, metastaticlesions associated with the aforementioned cancers. In some embodiments,the patient will have undergone one or more of surgical removal of atissue, chemotherapy, or other anti-cancer therapy and the primary orsole target will be metastatic lesions, e.g., metastases in the bone orlymph nodes or lung or liver or peritoneal cavity or the CNS or otherorgans.

The methods of the invention, e.g., methods of treatment, can furtherinclude the step of monitoring the subject, e.g., for a change (e.g., anincrease or decrease) in one or more of: tumor size; levels of a cancermarker, for a patient with cancer; the size or rate of appearance of newlesions, e.g., in a scan; the appearance of new disease-relatedsymptoms; the size of soft tissue mass, e.g., a decrease orstabilization; changes in blood flow measured by imaging technology;survival; progression-free survival; quality of life, e.g., amount ofdisease associated pain, e.g., bone pain; or any other parameter relatedto clinical outcome. In one embodiment, the subject can be monitored forbone marrow derived progenitor cell mobilization (e.g., EPC, HPC, iMC(MDSC) and/or MPC mobilization). The subject can be monitored in one ormore of the following periods: prior to beginning of treatment; duringthe treatment; or after one or more elements of the treatment have beenadministered. Monitoring can be used to evaluate the need for furthertreatment with the same preparation or for additional treatment withadditional agents. Generally, a decrease in one or more of theparameters described above is indicative of the improved condition ofthe subject.

The preparations described herein can be administered to a subject insingle or multiple doses to treat or prevent a metastatic or cancerousdisorder, e.g., a cancerous disorder described herein.

The preparations described herein can also be used to treatinflammatory, autoimmune, fibrotic, fibroproliferative, atopic, orangiogenic disorders. Examples of inflammatory disorders include but arenot limited to chronic obstructive pulmonary disease, asthma, rheumatoidarthritis, inflammatory bowel disease (including Crohns disease andulcerative colitis), multiple sclerosis, psoriasis, ischemia-reperfusioninjuries, septic shock, age-related macular degeneration (e.g., wetage-related macular degeneration), atherosclerosis, Alzheimer's disease,Parkinson's disease, cardiovascular disease, vasculitis, type I and IIdiabetes, metabolic syndrome, diabetic retinopathy, restenosis. Examplesof autoimmune diseases include but are not limited to asthma, rheumatoidarthritis, inflammatory bowel disease, multiple sclerosis, psoriasis,type I diabetes, systemic lupus erythematosus (SLE), Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, Guillain-Barré syndrome,autoimmune hepatitis, Myasthenia gravis. Examples of fibrotic diseasesinclude but are not limited to scleroderma, liver fibrosis, pancreaticfibrosis, chronic obstructive pulmonary disease, diabetic nephropathy,sarcoidosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis,neurofibromatosis, endometriosis, post-operative fibroids, restenosis.Examples of atopic disease include but are not limited to atopicdermatitis, atopic asthma, and allergic rhinitis.

Examples of fibroproliferative disorders include systemic and localscleroderma, keloids and hypertrophic scars, atherosclerosis,restenosis, fibrosarcoma, neurofibromatosis, and rheumatoid arthritis.Examples of scarring associated with trauma include scarring due tosurgery, chemotherapeutic-induced fibrosis, radiation-induced fibrosis,scarring associated with injury or burns.

In one embodiment, the polysaccharide preparations are used forinhibiting angiogenesis, e.g., to treat angiogenic disorders.Angiogenesis as used herein is the inappropriate formation of new bloodvessels. Angiogenic disorders include, but are not limited to, tumors,neovascular disorders of the eye, endometriosis, macular degeneration,osteoporosis, psoriasis, arthritis, cancer, hemangiomas, andcardiovascular disorders. It is understood that some disorders will fallwithin more than one category of disease described herein.

The preparations described herein can also be used to treat or preventinfectious disorders such as, e.g., malaria.

Combination Therapy

The methods and compositions of the invention can be used in combinationwith other therapeutic modalities. Administered “in combination”, asused herein, means that two (or more) different treatments are deliveredto the subject during the course of the subject's affliction with thedisorder, such that the effects of the treatments on the patient overlapat a point in time. In some embodiments, the delivery of one treatmentis still occurring when the delivery of the second begins, so that thereis overlap in terms of administration. This is sometimes referred toherein as “simultaneous” or “concurrent delivery.” In other embodiments,the delivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

In one embodiment, the methods of the invention include administering tothe subject a preparation described herein, in combination with one ormore additional therapies, e.g., surgery, radiation therapy, oradministration of another therapeutic preparation. In one embodiment,the additional therapy may include chemotherapy, e.g., a cytotoxicagent. In one embodiment the additional therapy may include a targetedtherapy, e.g. a tyrosine kinase inhibitor, a proteasome inhibitor, aprotease inhibitor. In one embodiment, the additional therapy mayinclude an anti-inflammatory, anti-angiogenic, anti-fibrotic, oranti-proliferative compound, e.g., a steroid, a biologicimmunomodulator, a monoclonal antibody, an antibody fragment, anaptamer, an siRNA, an antisense molecule, a fusion protein, a cytokine,a cytokine receptor, a bronchodialator, a statin, an anti-inflammatoryagent (e.g. methotrexate), an NSAID. In another embodiment, theadditional therapy could include combining therapeutics of differentclasses. The polysaccharide preparation and the additional therapy canbe administered simultaneously or sequentially.

Exemplary cytotoxic agents that can be administered in combination withthe polysaccharide preparation include antimicrotubule agents,topoisomerase inhibitors, antimetabolites, protein synthesis anddegradation inhibitors, mitotic inhibitors, alkylating agents,platinating agents, inhibitors of nucleic acid synthesis, histonedeacetylase and DNA methyltransferase inhibitors, nitrogen mustards,nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folateanalogs, nucleoside analogs, ribnucleotide reductase inhibitors, vincaalkaloids, taxanes, epothilones, intercalating agents, agents capable ofinterfering with a signal transduction pathway, agents that promoteapoptosis and radiation, antibody conjugates that bind surface proteinsto deliver a toxic agent. In one embodiment, the cytotoxic agent thatcan be administered with a preparation described herein is aplatinum-based agent (such as cisplatin), cyclophosphamide, dacarbazine,methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea,topotecan, irinotecan, azacytidine, vorinostat, ixabepilone, bortezomib,taxanes (paclitaxel, docetaxel), cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, vinorelbine, colchicin, anthracyclines (doxorubicin,epirubicin, daunorubicin), dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, adriamycin, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, ricin, and maytansinoids.

The combination therapy can also include a composition of the presentinvention coformulated with, and/or coadministered with, one or moreadditional therapeutic agents, e.g., one or more anti-cancer agents,cytotoxic or cytostatic agents, hormone treatment, small moleculeinhibitors of receptor tyrosine kinases and other tyrosine kinasesincluding HER-2, EGFR, VEGFR, BCR-ABL, c-KIT (such as Gefitinib,Erlotinib, Lapatinib, Sorafenib, Sunitinib, Imatinib, Dasatinib,Nilotinib) or mTOR (such as temsirolimus, everolimus, rapamycin), orcytokines or chemokines, vaccines, antibodies against cell membranereceptors pathways including EGF-EGFR, VEGF-VEGFR, CD19, CD20, CD3,CTLA-4 (such as Trastuzumab, Cetuximab, Panitumumab, Bevacizumab,Rituximab, Tositumomab) and/or other immunotherapies.

Anti-Angiogenic Agent or Tyrosine Kinase Inhibitors

The polysaccharide preparations described herein can be administered incombination with an anti-angiogenic agent or tyrosine kinase inhibitorto treat a subject having cancer, e.g., a primary tumor, or having or atrisk of having metastasis of a primary tumor. As discussed herein,administration of anti-angiogenic agents and tyrosine kinase inhibitorsto a subject having cancer is associated with mobilization of bonemarrow derived progenitor cells such as endothelial progenitor cells.

In one embodiment, the anti-angiogenic agent or tyrosine kinaseinhibitor is administered in an amount and/or dosing schedule that isassociated with (e.g., causes or results in) bone marrow derivedprogenitor cell mobilization. For example, the anti-angiogenic agent ortyrosine kinase inhibitor is administered in an amount and/or dosingschedule that is associated with (e.g., causes or results in)endothelial progenitor cell mobilization. The dose and/or dosingschedule can be a dose and/or dosing schedule described herein.

In one embodiment, the anti-angiogenic agent or tyrosine kinaseinhibitor is selected from the group consisting of: an epidermal growthfactor (EGF) pathway inhibitor (e.g., an epidermal growth factorreceptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF)pathway inhibitor (e.g., a vascular endothelial growth factor receptor(VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, aVEGFR-3 inhibitor)), a platelet derived growth factor (PDGF) pathwayinhibitor (e.g., a platelet derived growth factor receptor (PDGFR)inhibitor (e.g., a PDGFR-β inhibitor)), a TGF pathway inhibitor, a KITpathway inhibitor, a RAF-1 inhibitor and a RET inhibitor. In someembodiments, the subject has been treated or will be treated with ananti-angiogenic agent or a tyrosine kinase inhibitor selected from thegroup consisting of: bevacizumab (Avastin®), ranibizumab (Lucentis®),imatinib (Gleevec®), cetuximab (Erbitux®), sunitinib (Sutent®),sorafenib (Nexavar®), tivozanib (AV-951), cediranib (AZD2171), dasatinib(Sprycel®), nilotinib (AMN-107), CP-547632, erlotinib (Tarceva®),panitumumab (Vectibix®), pazopanib (Votrient®), axitinib and gefitinib(Iressa®).

A PDGF pathway inhibitor includes, without limitation, tyrphostin AG1296, tyrphostin 9,1,3-butadiene-1,1,3-tricarbonitrile,2-amino-4-(1H-indol-5-yl)-(9C1), imatinib (Gleevec®) and gefitinib(Iressa®) and those compounds generically and specifically disclosed inEuropean Patent No.: 0 564 409 and PCT Publication No.: WO 99/03854.

A VEGF pathway inhibitor includes, without limitation, anti-VEGFantibodies, e.g., bevacizumab (Avastin®), and small molecules, e.g.,sunitinib (Sutent®), sorafinib (Nexavar®), ZD6474 (also known asvandetanib) (Zactima™), SU6668, CP-547632 and AZD2171 (also known ascediranib) (Recentin™).

A EGF pathway inhibitor includes, without limitation, anti-EGFRantibodies, e.g., cetuximab (Erbitux®), panitumumab (Vectibix®), andgefitinib (Iressa®), and small molecules such as tyrphostin 46, EKB-569,erlotinib (Tarceva®), gefitinib (Iressa®), lapatinib (Tykerb®) and thosecompounds that are generically and specifically disclosed in WO97/02266, EP 0 564 409, WO 99/03854, EP 0 520 722, EP 0 566 226, EP 0787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO97/30034, WO 97/49688, WO 97/38983 and WO 96/33980.

In one embodiment, the cancer is gastrointestinal cancer. Thegastrointestinal cancer can be a chemotherapeutic refractory, achemotherapeutic resistant, and/or a relapsed cancer, e.g., thegastrointestinal cancer is refractory to imatinib mesylate, resistant toimatinib mesylate or relapsed after treatment with imatinib mesylate.

In an embodiment, the cancer is renal cell cancer, e.g., advanced ormetastatic renal cell carcinoma, e.g., a chemotherapeutic refractory, achemotherapeutic resistant, and/or a relapsed carcinoma, e.g., the renalcell carcinoma is refractory to a cytokine (e.g., interleukin-2 orinterferon), resistant to a cytokine (e.g., interleukin-2 or interferon)or relapsed after treatment with a cytokine (e.g., interleukin-2 orinterferon). In some embodiments, a renal cell cancer is treatedaccording to methods described herein with pazopanib (Votrient®) (e.g.,at a dose of 800 mg or less (e.g., 600 mg, 400 mg, 200 mg) daily, orsorafenib (Nexavar®) in combination with a polysaccharide preparationdescribed herein. In some embodiments, a polysaccharide described hereincan be administered in combination with sunitinib. In one embodiment,the sunitinib is administered orally at a dose of 20 mg, 25 mg, 30 mg,35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg sunitinib orally, once daily,on a schedule. In one embodiment, the schedule is administration ofsunitinib everyday for three, four or five weeks followed by one, two orthree weeks of no administration or continuously without ‘drug holiday’.

In an embodiment, the cancer is colorectal cancer, e.g., metastaticcolorectal cancer, e.g., a chemotherapeutic refractory, achemotherapeutic resistant, and/or a relapsed cancer. In someembodiments, a colorectal cell cancer is treated with a polysaccharidepreparation described herein in combination with bevacizumab (Avastin®)(e.g., at a dose of 5 to 10 mg/kg every 12, 13, 14, 15, 16 days), e.g.,in further combination with one or more of a topoisomerase inhibitor(e.g., topotecan, irinotecan, etoposide, teniposide, lamellarin D,camptothecin), a platinum-based agent (e.g., cisplatin, carboplatin,oxaliplatin), an antimetabolite (e.g., 5FU) and leucovorin.

In an embodiment, the cancer is lung cancer, e.g., small cell lungcancer or non-small cell lung cancer, e.g., a chemotherapeuticrefractory, a chemotherapeutic resistant, and/or a relapsed cancer. Insome embodiments, the lung cell cancer is treated with a polysaccharidepreparation described herein in combination with bevacizumab (Avastin®)(e.g., at a dose of 5 to 10 mg/kg every 12, 13, 14, 15, 16 days), e.g.,in further combination with one or more of a topoisomerase inhibitor(e.g., topotecan, irinotecan, etoposide, teniposide, lamellarin D,camptothecin), a platinum-based agent (e.g., cisplatin, carboplatin,oxaliplatin), an antimetabolite (e.g., 5FU) and leucovorin.

In an embodiment, the cancer is breast cancer, e.g., metastatic breastcancer, e.g., a chemotherapeutic refractory, a chemotherapeuticresistant, and/or a relapsed breast cancer. The breast cancer can beestrogen receptor positive breast cancer; estrogen receptor negativebreast cancer; HER-2 positive breast cancer; HER-2 negative breastcancer; progesterone receptor positive breast cancer; progesteronereceptor negative breast cancer; estrogen receptor negative, HER-2negative and progesterone receptor negative breast cancer (i.e., triplenegative breast cancer). In some embodiments, the breast cancer istreated with a polysaccharide preparation described herein incombination with bevacizumab (Avastin®) (e.g., at a dose of 5 to 10mg/kg every 12, 13, 14, 15, 16 days), e.g., in further combination witha taxane (e.g., docetaxel, paclitaxel, larotaxel, cabizitaxel). Also,e.g., in combination with anthracycline (daunorubicin (Daunomycin®),doxorubicin (Adriamycin®)), e.g. in combination with platinum (e.g.cisplatin) e.g., in combination with estrogen inhibitor (e.g. aromataseinhibitors, tamoxifen (Nolvadex®), exemestane (Aromasin®), anastrozole(Arimidex®) and letrozole (Femara®), e.g. in combination with EGF/HER2inhibitors (e.g. Lapatinib (Tykerb®), trastuzumab (Herceptin®).

In an embodiment, the cancer is a glioblastoma, e.g., a chemotherapeuticrefractory, a chemotherapeutic resistant, and/or a relapsedglioblastoma. In certain embodiments, the glioblastoma is treated with apolysaccharide preparation described herein in combination withbevacizumab (Avastin®) (e.g., at a dose of 5 to 10 mg/kg every 12, 13,14, 15, 16 days).

In one embodiment, the cancer is gastrointestinal cancer and apolysaccharide described herein is administered in combination withsunitinib. In some embodiments, the sunitinib is administered orally ata dose of 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45, mg, 50 mg, 55 mg, 60mg, once daily, on a schedule. In one embodiment, the schedule isadministration of sunitinib everyday for three, four or five weeksfollowed by one, two or three weeks of no administration or continuouslywithout ‘drug holiday’.

In an embodiment, the cancer is a leukemia (e.g., chronic myeloidleukemia or acute lymphoblastic leukemia, e.g., Philadelphia chromosomepositive chronic myeloid leukemia or acute lymphoblastic leukemia),e.g., a chemotherapeutic refractory, a chemotherapeutic resistant,and/or a relapsed leukemia, e.g., refractory, resistant, and/or relapsedto imatinib. In some embodiments, the leukemia is treated with apolysaccharide preparation described herein in combination withdasatinib (e.g., at a dose of 120 mg/day, 130 mg/day, 140 mg/day, 150mg/day, e.g., administered twice daily).

In an embodiment, the cancer is a pancreatic cancer (e.g., advancedpancreatic cancer). In some embodiments, the pancreatic cancer istreated with a polysaccharide preparation described herein incombination with gemcitabine, erlotinib, Abraxane (a taxol conjugate), amTOR inhibitor, a VEGF inhibitor (e.g., a VEGF inhibitor describedherein), a sonic hedgehog inhibitor.

Vascular Disrupting Agents

The polysaccharide preparations described herein can be administered incombination with a vascular disrupting agent to treat a subject havingcancer, e.g., a primary tumor, or having or at risk of having metastasisof a primary tumor. The administration of vascular disrupting agents isassociated with mobilization of bone marrow derived progenitor cellssuch as endothelial progenitor cells in subjects having cancer.

In one embodiment, the vascular disrupting agent is administered in anamount and/or dosing schedule that is associated with (e.g., causes orresults in) bone marrow derived progenitor cell mobilization. Forexample, the vascular disrupting agent is administered in an amountand/or dosing schedule that is associated with (e.g., causes or resultsin) endothelial progenitor cell mobilization. The dose and/or dosingschedule can be a dose and/or dosing schedule described herein.

Exemplary vascular disrupting agents include, but are not limited to,AVE8062, vadimezan, ZD6126, combretastatin A-4 disodium phosphate (CA4P)or Oxi4503, DMXAA (ASA404), NPI-2358.

In one embodiment, the cancer is lung cancer (e.g., small cell lungcancer or non-small cell lung cancer). The lung cancer can be resistant,relapsed or refractory to treatment with a chemotherapeutic agent, e.g.,a VEGF pathway inhibitor (e.g., bevacizumab) or an EGF pathwayinhibitor. The lung cancer can be locally advanced or metastatic lungcancer. In another embodiment, the cancer is urothelial cancer (e.g.,cancer of the bladder, urethra, ureter, renal pelvis), e.g., locallyadvanced or metastatic urothelial cancer. The urothelial cancer can beresistant, relapsed or refractory to another chemotherapeutic agent,e.g., a platinum based agent (e.g., cisplatin, carboplatin, oxaliplatin)or a pyrimidine analog (e.g., gemcitabine). A polysaccharide describedherein can be administered in combination with ASA404, e.g., ASA404 at adose of 1,600 mg/m², 1,700 mg/m², 1,800 mg/m², 1,900 mg/m², 2,000 mg/m²on a schedule. In one embodiment, the schedule is administration ofASA404 every 18, 19 20, 21, 22, 23 or 24 days, e.g., for 4, 5, 6, 7cycles. The treatment can further include administration of one or moreadditional chemotherapeutic agent, e.g., a taxane (e.g., docetaxel,paclitaxel, larotaxel, cabizitaxel) or a platinum based agent (e.g.,cisplatin, carboplatin, oxaliplatin). In some embodiments, the lungcancer is treated with a polysaccharide preparation described herein incombination with NPI-2358 (e.g., at a dose of 20, 30, 40 mg/m²).

In an embodiment, the cancer is a head and neck cancer (e.g., anaplasticcarcinoma of the thyroid), e.g., locally advanced or metastatic head andneck cancer. In another embodiment, the cancer is a glioma. In yetanother embodiment, the cancer is lung cancer (e.g., small cell lungcancer or non small cell lung cancer), e.g., locally advanced ormetastatic lung cancer. The cancer can be chemotherapeutic refractory, achemotherapeutic resistant, and/or a relapsed. In certain embodiments,the cancer is treated with a polysaccharide preparation described hereinin combination with CA4P (e.g., at a dose of 50 mg/m², 60 mg/m², 70mg/m² on a schedule. The dosing schedule can be, e.g., administration ofCA4P weekly for three weeks then one week without administration.

In an embodiment, the cancer is a sarcoma (e.g., a soft tissue sarcoma),e.g., locally advanced or metastatic sarcoma. The cancer can bechemotherapeutic refractory, a chemotherapeutic resistant, and/or arelapsed to another chemotherapeutic agent, e.g., an anthracycline or analkylating agent (e.g., ifosfamide). In certain embodiments, the canceris treated with a polysaccharide preparation described herein incombination with AVE8026 (e.g., at a dose of 15 mg/m², 20 mg/m², 25mg/m², 30 mg/m² on a schedule). The dosing schedule can be, e.g.,administration of AVE8026 every three weeks. In some embodiments, thetreatment can further include administration of one or more additionalchemotherapeutic agents, e.g., a platinum based agent (e.g., cisplatin,carboplatin, oxaliplatin) and a taxane (e.g., docetaxel, paclitaxel,larotaxel, cabizitaxel).

Taxanes

The polysaccharide preparations described herein can be administered incombination with a taxane to treat a subject having cancer, e.g., aprimary tumor, or having or at risk of having metastasis of a primarytumor. As discussed herein, administration of a taxane to a subjecthaving cancer is associated with mobilization of bone marrow derivedprogenitor cells such as endothelial progenitor cells.

In one embodiment, the taxane is administered in an amount and/or dosingschedule that is associated with (e.g., causes or results in) bonemarrow derived progenitor cell mobilization. For example, the taxane isadministered in an amount and/or dosing schedule that is associated with(e.g., causes or results in) endothelial progenitor cell mobilization.The dose and/or dosing schedule can be a dose and/or dosing scheduledescribed herein.

In one embodiment, the cancer is breast cancer (e.g., locally advancedor metastatic breast cancer). The breast cancer can be estrogen receptorpositive breast cancer; estrogen receptor negative breast cancer; HER-2positive breast cancer; HER-2 negative breast cancer; progesteronereceptor positive breast cancer; progesterone receptor negative breastcancer; estrogen receptor negative, HER-2 negative and progesteronereceptor negative breast cancer (i.e., triple negative breast cancer).The breast cancer can be resistant, relapsed or refractory to treatmentwith a chemotherapeutic agent, e.g., an alkylating agent (e.g.,cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide) oran anthracycline (e.g., daunorubicin, doxorubicin, epirubicin,valrubicin and idarubicin). In some embodiments, a polysaccharidedescribed herein can be administered in combination with docetaxel,e.g., docetaxel at a dose of 60 mg/m², 70 mg/m², 75 mg/m², 80 mg/m², 90mg/m², 100 mg/m², 105 mg/m², 110 mg/m², 115 mg/m² on a schedule. In oneembodiment, the schedule is administration of docetaxel every threeweeks. In another embodiment, a polysaccharide described herein can beadministered in combination with paclitaxel, e.g., paclitaxel at a doseof 125 mg/m², 135 mg/m², 145 mg/m², e.g., infused over about 2, 3, or 4hours, or 165 mg/m², 175 mg/m², 185 mg/m², 195 mg/m², e.g., infused overabout 22, 23, 24 or 25 hours, on a schedule. In one embodiment, theschedule is administration of paclitaxel every three weeks. Thetreatment can further include administration of one or more additionalchemotherapeutic agent, e.g., a vinca alkaloid (e.g., vinblastine,vincristine, vindesine, vinorelbine) or an anthracycline (e.g.,daunorubicin, doxorubicin, epirubicin, valrubicin and idarubicin, or aplatinum based agent (e.g. cisplatin).

In another embodiment, the cancer is lung cancer (e.g., small cell lungcancer or non small cell lung cancer), e.g., locally advanced ormetastatic lung cancer. The lung cancer can be resistant, relapsed orrefractory to a chemotherapeutic agent, e.g., a platinum based agent(e.g., cisplatin, carboplatin, oxaliplatin). A polysaccharide describedherein can be administered in combination with docetaxel, e.g.,docetaxel at a dose of 60 mg/m², 70 mg/m², 75 mg/m², 80 mg/m², 90 mg/m²,100 mg/m², 105 mg/m², 110 mg/m², 115 mg/m² on a schedule. In oneembodiment, the schedule is administration of docetaxel every threeweeks. In another embodiment, a polysaccharide described herein can beadministered in combination with paclitaxel, e.g., paclitaxel at a doseof 125 mg/m², 135 mg/m², 145 mg/m², e.g., infused over about 2, 3, or 4hours, or 165 mg/m², 175 mg/m², 185 mg/m², 195 mg/m², e.g., infused overabout 22, 23, 24 or 25 hours, on a schedule. In one embodiment, theschedule is administration of paclitaxel every three weeks. Thetreatment can further include administration of one or more additionalchemotherapeutic agent, e.g., a vinca alkaloid (e.g., vinblastine,vincristine, vindesine, vinorelbine) or an alkylating agent (e.g.,cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide).

In one embodiment, the cancer is prostate cancer (e.g., locally advancedor metastatic prostate cancer). The prostate cancer can be resistant,relapsed or refractory to treatment with a chemotherapeutic agent. Apolysaccharide described herein can be administered in combination withdocetaxel, e.g., docetaxel at a dose of 60 m g/m², 70 mg/m², 75 mg/m²,80 mg/m², 90 mg/m², 100 mg/m², 105 mg/m², 110 mg/m², 115 mg/m² on aschedule. In one embodiment, the schedule is administration of docetaxelevery three weeks. In another embodiment, a polysaccharide describedherein can be administered in combination with docetaxel, e.g.,docetaxel at a dose of 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40 mg/m²on a schedule. In one embodiment, the schedule is weekly administrationof docetaxel. The treatment can further include administration of one ormore additional chemotherapeutic agent.

In one embodiment, the cancer is ovarian cancer (e.g., locally advancedor metastatic ovarian cancer). The ovarian cancer can be resistant,relapsed or refractory to treatment with a chemotherapeutic agent, e.g.,a platinum based agent (e.g., cisplatin, carboplatin, oxaliplatin). Apolysaccharide described herein can be administered in combination withpaclitaxel, e.g., paclitaxel at a dose of 125 mg/m², 135 mg/m², 145mg/m², e.g., infused over about 2, 3, or 4 hours, or 165 mg/m², 175mg/m², 185 mg/m², 195 mg/m², e.g., infused over about 22, 23, 24 or 25hours, on a schedule. In one embodiment, the schedule is administrationof paclitaxel every three weeks. The treatment can further includeadministration of one or more additional chemotherapeutic agent.

In one embodiment, the cancer is a sarcoma (e.g., AIDS-related Kaposisarcoma), e.g., locally advanced or metastatic sarcoma). The sarcoma canbe resistant, relapsed or refractory to treatment with achemotherapeutic agent, e.g., an anthracycline (e.g., daunorubicin,doxorubicin, epirubicin, valrubicin and idarubicin). A polysaccharidedescribed herein can be administered in combination with paclitaxel,e.g., paclitaxel at a dose of 125 mg/m², 135 mg/m², 145 mg/m², e.g.,infused over about 2, 3, or 4 hours, or 155 mg/m², 165 mg/m², 175 mg/m²,185 mg/m², 195 mg/m², e.g., infused over about 22, 23, 24 or 25 hours,on a schedule. In one embodiment, the schedule is administration ofpaclitaxel every three weeks. The treatment can further includeadministration of one or more additional chemotherapeutic agent.

Pyrimidine Analogues

The polysaccharide preparations described herein can be administered incombination with a pyrimidine analogue (e.g., fluorouracil) to treat asubject having cancer, e.g., a primary tumor, or having or at risk ofhaving metastasis of a primary tumor. The administration of pyrimidineanalogues such as fluorouracil can be associated with mobilization ofbone marrow derived progenitor cells such as endothelial progenitorcells in subjects having cancer.

In one embodiment, the pyrimidine analogue (e.g., fluorouracil) isadministered in an amount and/or dosing schedule that is associated with(e.g., causes or results in) bone marrow derived progenitor cellmobilization. For example, the vascular disrupting agent is administeredin an amount and/or dosing schedule that is associated with (e.g.,causes or results in) endothelial progenitor cell mobilization. The doseand/or dosing schedule can be a dose and/or dosing schedule describedherein.

In one embodiment, the cancer is breast cancer (e.g., locally advancedor metastatic breast cancer). The breast cancer can be estrogen receptorpositive breast cancer; estrogen receptor negative breast cancer; HER-2positive breast cancer; HER-2 negative breast cancer; progesteronereceptor positive breast cancer; progesterone receptor negative breastcancer; estrogen receptor negative, HER-2 negative and progesteronereceptor negative breast cancer (i.e., triple negative breast cancer).The breast cancer can be resistant, relapsed or refractory to treatmentwith a chemotherapeutic agent, e.g., an alkylating agent (e.g.,cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), ananthracycline (e.g., daunorubicin, doxorubicin, epirubicin, valrubicinand idarubicin), a taxane (e.g., docetaxel or paclitaxel) or a platinumbased agent (e.g. cisplatin). In some embodiments, a polysaccharidedescribed herein can be administered in combination with fluorouracil,e.g., fluorouracil at a dose of 8 mg/m², 10 mg/m², 12 mg/m², 14 mg/m²,16 mg/m² on a schedule. In one embodiment, the schedule isadministration of fluorouracil once daily for four days, and then, e.g.,at a reduced dose on day 6, 8, 10 and 12. The treatment can furtherinclude administration of one or more additional chemotherapeutic agent,e.g., an alkylating agent (e.g., cyclophosphamide, dacarbazine,melphalan, ifosfamide, temozolomide), an anthracycline (e.g.,daunorubicin, doxorubicin, epirubicin, valrubicin and idarubicin), or ataxane (e.g., docetaxel or paclitaxel). The treatment can furtherinclude administration of leucovorin.

In one embodiment, the cancer is colorectal cancer (e.g., locallyadvanced or metastatic colorectal cancer). The colorectal cancer can beresistant, relapsed or refractory to treatment with a chemotherapeuticagent. In some embodiments, a polysaccharide described herein can beadministered in combination with fluorouracil, e.g., fluorouracil at adose of 8 mg/m², 10 mg/m², 12 mg/m², 14 mg/m², 16 mg/m² on a schedule.In one embodiment, the schedule is administration of fluorouracil oncedaily for four days, and then, e.g., at a reduced dose on day 6, 8, 10and 12. The treatment can further include administration of one or moreadditional chemotherapeutic agent, e.g., an alkylating agent (e.g.,cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), ananthracycline (e.g., daunorubicin, doxorubicin, epirubicin, valrubicinand idarubicin), or a taxane (e.g., docetaxel, paclitaxel, larotaxel,cabizitaxel). The treatment can further include administration ofleucovorin.

In one embodiment, the cancer is gastric cancer (e.g., locally advancedor metastatic gastric cancer). The gastric cancer can be resistant,relapsed or refractory to treatment with a chemotherapeutic agent. Insome embodiments, a polysaccharide described herein can be administeredin combination with fluorouracil, e.g., fluorouracil at a dose of 8mg/m², 10 mg/m², 12 mg/m², 14 mg/m², 16 mg/m² on a schedule. In oneembodiment, the schedule is administration of fluorouracil once dailyfor four days, and then, e.g., at a reduced dose on day 6, 8, 10 and 12.The treatment can further include administration of one or moreadditional chemotherapeutic agent, e.g., a platinum based agent (e.g.,cisplatin, carboplatin, oxaliplatin), a taxane (docetaxel, paclitaxel,larotaxel, cabizitaxel) and an anthracycline (e.g., daunorubicin,doxorubicin, epirubicin, valrubicin and idarubicin). The treatment canfurther include administration of leucovorin.

In one embodiment, the cancer is pancreatic cancer (e.g., locallyadvanced or metastatic pancreatic cancer). The pancreatic cancer can beresistant, relapsed or refractory to treatment with a chemotherapeuticagent. In some embodiments, a polysaccharide described herein can beadministered in combination with fluorouracil, e.g., fluorouracil at adose of 8 mg/m², 10 mg/m², 12 mg/m², 14 mg/m², 16 mg/m² on a schedule.In one embodiment, the schedule is administration of fluorouracil oncedaily for four days, and then, e.g., at a reduced dose on day 6, 8, 10and 12. The treatment can further include administration of one or moreadditional chemotherapeutic agent. The treatment can further includeadministration of leucovorin.

Growth Factors for Blood Cells

The polysaccharide preparations described herein can be administered incombination with a chemotherapeutic agent that is administered incombination with growth factors for blood cells (e.g. myeloid cells,granulocytes, and red blood cells) to treat a subject having cancer,e.g., a primary tumor, or having or at risk of having metastasis of aprimary tumor. The administration of a chemotherapeutic agent thatrequires co administration of a growth factor for blood cells (e.g.myeloid cells and red blood cells), e.g., to counter one or more sideeffect of the chemotherapeutic agent, may be associated withmobilization of bone marrow derived progenitor cells such as endothelialprogenitor cells in subjects having cancer.

In one embodiment, the method includes administering thechemotherapeutic agent in combination with a growth factor and thensubsequent administration of a polysaccharide preparation describedherein. For example, the polysaccharide preparation can be administeredone, two, three, five, ten, fifteen, twenty hours, or 1, 2, 3, 4 daysafter the administration of the growth factor.

Exemplary growth factors include, but are not limited to, colonystimulating factors (e.g., granulocyte colony stimulating factor (GCSF),granulocyte macrophage colony stimulating factor (GM-CSF)), anderythropoietin.

In one embodiment, the subject has one of the following cancers: lungcancer (e.g., small cell lung cancer or non small cell lung cancer),urothelial cancer, a nonmyeloid malignancy, breast cancer, ovariancancer and a neuroblastoma.

In one embodiment, the subject has lung cancer (e.g., small cell lungcancer or non small cell lung cancer) and the method includesadministering an anthracycline (e.g., doxorubicin, daunorubicin,epirubicin, idarubicin, mitoxantrone, valrubicin), a topoisomeraseinhibitor (e.g., topotecan, irinotecan, etoposide, teniposide,lamellarin D, SN-38, camptothecin) and/or an alkylating agent (e.g.,cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide) incombination with a growth factor (e.g., a colony stimulating factor,e.g., GCSF, GM-CSF), and then subsequently administering apolysaccharide preparation described herein.

In one embodiment, the subject has urothelial cancer and the methodincludes administering an anthracycline (e.g., doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), atopoisomerase inhibitor (e.g., topotecan, irinotecan, etoposide,teniposide, lamellarin D, SN-38, camptothecin) and/or an alkylatingagent (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide,temozolomide) in combination with a growth factor (e.g., a colonystimulating factor, e.g., GCSF, GM-CSF), in combination with apolysaccharide preparation described herein, e.g., concomitantly orserially.

In one embodiment, the subject has a nonmyeloid cancer and the methodincludes administering an anthracycline (e.g., doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), aplatinum based agent (e.g., cisplatin, carboplatin, oxaliplatin), avinca alkaloid (e.g., vinblastine, vincristine, vindesine andvinorelbine) and/or an antimetabolite (e.g., methotrexate) incombination with a growth factor (e.g., a colony stimulating factor,e.g., GCSF, GM-CSF), in combination with a polysaccharide preparationdescribed herein, e.g., concomitantly or serially.

In one embodiment, the subject has breast cancer or ovarian cancer andthe method includes administering a platinum based agent (e.g.,cisplatin, carboplatin, oxaliplatin), a topoisomerase inhibitor (e.g.,topotecan, irinotecan, etoposide, teniposide, lamellarin D, SN-38,camptothecin) and/or an alkylating agent (e.g., cyclophosphamide,dacarbazine, melphalan, ifosfamide, temozolomide) in combination with agrowth factor (e.g., a colony stimulating factor, e.g., GCSF, GM-CSF),in combination with a polysaccharide preparation described herein, e.g.,concomitantly or serially. The breast cancer can be estrogen receptorpositive breast cancer; estrogen receptor negative breast cancer; HER-2positive breast cancer; HER-2 negative breast cancer; progesteronereceptor positive breast cancer; progesterone receptor negative breastcancer; estrogen receptor negative, HER-2 negative and progesteronereceptor negative breast cancer (i.e., triple negative breast cancer).

In one embodiment, the subject has a neuroblastoma and the methodincludes administering an anthracycline (e.g., doxorubicin,daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), aplatinum based agent (e.g., cisplatin, carboplatin, oxaliplatin) and/oran alkylating agent (e.g., cyclophosphamide, dacarbazine, melphalan,ifosfamide, temozolomide) in combination with a growth factor (e.g., acolony stimulating factor, e.g., GCSF, GM-CSF), and then subsequentlyadministering a polysaccharide preparation described herein.

Radiation and Surgery

The polysaccharide preparations described herein can be administered incombination with radiation therapy or surgery to treat a subject havingcancer, e.g., a primary tumor, or having or at risk of having metastasisof a primary tumor. As discussed herein, administration of surgeryand/or radiation to a subject having cancer is associated withmobilization of bone marrow derived progenitor cells such as EPCs.

Other Embodiments

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Preparation of a Polysaccharide Preparation

This example describes the production of a polysaccharide preparationdescribed herein.

Overview:

Glycol Split low molecular weight heparin alcohol (GS-LMWH—CH₂—OH) wasgenerated from unfractionated heparin (UFH) by controlled nitrous aciddepolymerization followed by oxidative glycol-splitting and subsequentreduction to an alcohol. In the first step, UFH was depolymerized toobtain depolymerized heparin (DPH—CHO) having an anhydromannose moietyat the reducing end of the polysaccharide. This was followed by Step IIoxidative cleavage of the 2,3-diols present in the depolymerized heparinwith sodium periodate to generate ring opened glycol split residuesalong the heparin chain (GS-DPH—CHO). The Step III involved a reductionstep, wherein the aldehydic moieties are converted to alcohols usingsodium borohydride to generate Glycol Split low molecular weight heparinalcohol.

Method Overview:

The following paragraphs describe the preparation and properties of apolysaccharide preparation described herein.

1. Depolymerization:

Unfractionated Heparin (10 g) was dissolved in 100 mL of de-ionizedwater equilibrated at room temperature. The pH of this solution wassubsequently lowered to pH 3.1, following which sodium nitrite (0.03 M)was added. This reaction solution was allowed to stir for 3 hoursfollowing which the pH was neutralized prior to addition of sodiumchloride (10 g). After complete dissolution of salt, methanol (200 mL)was added to this solution with constant stirring. The precipitateobtained was then aged at 6° C. for 2 hours. This precipitate was thenfiltered and dried to obtain DPH in 80-85% yield and possessing thefollowing characteristics:

Mw: 5300-6100

Mw Distribution: (i) <3000 Daltons: 23-30%

-   -   (ii) 3000-8000 Daltons: 50-55%    -   (iii) >8000 Daltons: 15-22%

Anti-Xa Activity: 80-120 IU/mg

Anti-IIa Activity: 40-70 IU/mg

2. Periodate Oxidation

The aldehyde (5 g) obtained in Step I was dissolved in 50 mL waterequilibrated at 5° C. To this solution was added cooled NaIO₄ solution(0.1M, 50 mL) and the reaction mixture was allowed to stir in theabsence of light for 16 hours. On completion, the reaction was quenchedby the addition of diethylene glycol (10 mL), following which thetemperature was raised back to room temperature. Five grams of sodiumchloride was then added to this solution, followed by addition of 150 mLmethanol to precipitate the heparin. The precipitate was allowed to ageat 6° C. for 2 hours before filtration and drying to yield aglycol-split polysaccharide (95-98% yield) with the followingcharacteristics:

Mw: 5000-5800

Mw Distribution: (i) <3000 Daltons: 25-30%

-   -   (ii) 3000-8000 Daltons: 55-60%    -   (iii) >8000 Daltons: 15-20%

3. Reduction

The glycol split polysaccharide (4 g) obtained above in Step II wasdissolved in 40 mL water maintained at 5° C. To this solution was addedsodium borohydride (0.4 g) and the reaction mixture subsequently stirredfor 1 hour. After 1 hour, the reaction mixture was brought to roomtemperature, followed by the addition of sodium chloride (4 g).Following salt dissolution, methanol (80 mL) was added to this solutionaccompanied with constant stirring. The precipitate thus obtained wasthen allowed to age at 6° C. for 2 hours before filtration and drying toyield the desired product. A polysaccharide preparation with thefollowing characteristics was thus obtained in 55-60% yield:

Mw: 5500-6200

Mw Distribution: (i) <3000 Daltons: 17-23%

-   -   (ii) 3000-8000 Daltons: 56-62%    -   (iii) >8000 Daltons: 17-22%

Anti-Xa Activity: 1-20 IU/mg

Anti-IIa Activity: 1-10 IU/mg

Example 2 Preparation of a Polysaccharide Preparation Depolymerization:

Unfractionated Heparin is dissolved in 10-fold volume of de-ionizedwater equilibrated at room temperature. The pH of this solution isadjusted to pH 3.1, and sodium nitrite (0.03 M) is added. This reactionsolution is allowed to stir for several hours following which the pH isneutralized prior to addition of sodium chloride (same amount asstarting UFH material). After complete dissolution of salt, at least 2volumes of methanol are added to this solution with constant stirring.The precipitate obtained is aged at about room temperature for about 1hour. This precipitate is then filtered and dried to obtain DPH in80-85% typical yield.

Periodate Oxidation:

The aldehyde obtained in Step I is dissolved in about 10 volumes ofwater equilibrated at 5° C. To this solution is added an equal volume ofcooled NaIO₄ solution (0.1 M) and the reaction mixture is allowed tostir for 16 hours. On completion, the reaction is quenched by theaddition of an alcohol, following which the temperature is raised backto room temperature. Sodium chloride (double the amount as startingaldehyde material) is then added to this solution, followed by additionof at least 3 volumes methanol to precipitate the heparin. Theprecipitate is allowed to age at about room temperature for 2 hoursbefore filtration and drying to yield a glycol-split polysaccharide(typically about 95-98% yield).

Reduction:

The glycol split polysaccharide obtained in Step II is dissolved in 10volumes of water maintained at 5° C. To this solution is added sodiumborohydride (one tenth the starting amount of GS polysaccharide) and thereaction mixture is subsequently stirred for 1 hour. The reactionmixture is then brought to room temperature, followed by the addition ofsodium chloride (same amount as the starting amount of GSpolysaccharide). Following salt dissolution, 2 volumes of methanol isadded to this solution accompanied with constant stirring. Theprecipitate thus obtained is allowed to age at about room temperaturebefore filtration and drying to yield the desired product. A MONC402LMWH-sodium preparation with the following characteristics is thusobtained in approximately 55-60% yield:

Mw: 5000-7800 Daltons

Mw Distribution: (i) <3000 Daltons: 15-25%

-   -   (ii) 3000-8000 Daltons: 55-65%    -   (iii) >8000 Daltons: 15-25%

Anti-Xa Activity: 1-20 IU/mg

Anti-IIa Activity: 1-20 IU/mg

Example 3 Anti-Metastatic Properties of Polysaccharide Preparations

This example shows that the polysaccharide preparations have anti-cancerand anti-metastatic activity in multiple models of metastasis.

Model A: Murine Melanoma Experimental Metastasis (B16F10 iv) Model

A polysaccharide preparation produced as described in Example 1 (hereinreferred to as “MONC402”) showed anti-metastasis activity in a murinemelanoma experimental metastasis model.

Female C57BL/6 mice (9-10 weeks old) were treated once with a singledose (10 mg/kg) of MONC402, dalteparin/Fragmin® (a LMWH which has beenreported to decrease metastasis), or MONC 202 (negative control,N-desulfated polysaccharide) immediately before i.v. injection of 2×10⁵B16F10 cells. Mice were sacrificed on day 21 and tumor burden wascalculated as lung weight-normal lung weight. As shown in FIG. 1,MONC402 significantly inhibited B16F10 colonization of the lung relativeto a pooled (untreated) control.

Model B: Colon Cancer Metastasis to the Liver

MONC402 showed prophylactic anti-metastasis activity in an orthotopicliver metastasis model.

Liver metastasis was initiated by intraperitoneal injection of C170HM2human colorectal tumor cells into male MF1 nude (nu/nu) athymic mice.5FU/leucovorin was used as a positive control.

C170HM2 cells were maintained in vitro in RPMI culture medium (Sigma)containing 10% (v/v) heat inactivated fetal bovine serum and 2 mML-glutamine at 37° C. in 5% CO₂ and humidified conditions. Cells fromsub-confluent monolayers were harvested with 0.025% EDTA, washed inculture medium and re-suspended in sterile phosphate buffered saline, pH7.4 (PBS) for in vivo administration. 1.5×10⁶ cells in a volume of 1 mlwere injected intraperitoneally into 65 mice, and the mice wereallocated into treatment groups as below.

Group 1: n = 10 Vehicle control Group 2: n = 10 25 mg/kg 5FU/leucovorini.v. cycled on days 1, 3, 5, 7 Group 3: n = 10  5 mg/kg compound 1(Dalteparin) s.c. once daily Group 4: n = 10  5 mg/kg compound 2(MONC402) s.c. once daily Group 5: n = 10 15 mg/kg compound 2 s.c. oncedaily Group 6: n = 10 30 mg/kg compound 2 s.c. once daily Group 7: n = 5Untreated

Treatment was initiated on day 1 following cell injection and continueduntil day 35 or until the clinical condition of the animal requiredtermination. Groups 5 and 6 missed one dose on day 5. No adverse affectsof the test compounds in mice bearing the tumors were observed.

The study was terminated on day 35, and the tumors in the liver wereexcised and weighed. The numbers of lung nodules are also counted. Themean liver tumor weights and cross-sectional area are summarized inTable 1.

TABLE 1 C170HM2 model: summary of mean liver tumor weight andstatistical analysis Mean tumor weight Mean tumor area (% of One way (%of One way Group Treatment (g) vehicle) ANOVA (mm²) vehicle) ANOVA 1Vehicle 0.097 100.00 — 34.18 100.00 — 2 5FU/Leu 0.037 11.94 p = 0.00613.12 15.7 p = 0.011 3  5 mg/kg Dalteparin 0.018 18.56 p = 0.017 8.0923/67 p = 0.031 4  5 mg/kg MONC402 0.057 58.76 NS 18.34 53.66 NS 5 15mg/kg 0.010 10.31 p = 0.007 6.95 20.33 p = 0.016 MONC402 6 30 mg/kg0.003 3.09 p = 0.004 0.96 2.80 p = 0.004 MONC402 7 Untreated control0.31 — p = 0.035 83.58 244.53 p = 0.084 NS = not significant

Both 15 mg/kg and 30 mg/kg MONC402 significantly reduced the liver tumorsize by 90% (p=0.007) and 97% (p=0.004) respectively and also weresignificantly more effective than 5FU/leucovorin (p=0.041 and p=0.011,respectively). Dalteparin (group 3) reduced liver tumor weight byapproximately 81% (p=0.017) when compared to the vehicle control group.Similarly, the cross-sectional area of the tumors also showedsignificant reduction with dalteparin (p=0.027) and 15 and 30 mg/kgMONC402 (p=0.016 and p=0.004, respectively).

Mouse weights were monitored for the duration of the study. The mouseweights for each group remained within an acceptable range for allgroups throughout the study.

Model C: Breast Cancer Metastasis to the Lung

MONC402 also showed anti-metastasis activity in a syngeneic orthotopicmodel of breast cancer metastasis (4T1).

Female BALB/c mice 8 weeks of age (WOA) were injected with 8×10⁴ 4T1cell intra mammary fat pad. Daily treatment with saline or MONC402 withor without weekly treatment of cisplatin started on day 5. Primarytumors were removed on day 9 and weighed.

As shown in Table 2, cisplatin combined with MONC402 (10, 20, 30 mg/kg)showed a statistically significant decrease in lung metastasis comparedto saline control group as determined by lung weight and tumor nodulecounting (p<0.05, One way ANOVA). Combination therapy groups(Cisplatin+MONC402 10/20/30 mg/kg) also had lower incidence of mammarytumor regrowth, thoracic cavity tumor metastasis, and weight loss (>2 g)in the last 3 days before termination of the experiment. Combinationtherapy groups had higher incidence of transient weight loss (>2 g) theweek after surgery but recovered in one week.

TABLE 2 4T1 model: macroscopic tumor metastasis counts Lung tumorAverage Total Total nodule #/ lung tumor tumor Average tumor groups # ofmice animal size nodule tumor size volume Saline 15 6.0 ± 4.7 1.4 90 2.0122.10 Cisplatin 16 5.6 ± 4.4 1.41 89 1.36 134.13 MONC402 30 mg/kg 168.4 ± 7.0 1.19 135 1.11 140.98 Cisplatin + MONC402 16 3.1 ± 3.7 0.88 490.85 28.78 30 mg/kg Cisplatin + MONC402 15 2.3 ± 2.9 0.8 34 1.0 18.66 20mg/kg Cisplatin + MONC402 16 2.3 ± 2.5 1.41 37 1.41 57.39 10 mg/kguntreated 7 12.9 ± 14.0 0.98 90 1.3 65.06

In a second 4T1 experiment, female BALB/c mice 8 WOA were injected with8×10⁴ 4T1 cells intra mammary fat pad. Continuous osmotic pump deliveryof saline or MONC 402 with weekly treatment of saline or Cisplatinstarted on day 4. Primary tumors were removed on day 9. There were nosignificant differences between the groups in primary tumor weight.However, immunohistology analyses showed significant decrease inmicrovessel density in tumors from mice treated with the combination ofCisplatin and M-ONC 402. The experiment terminated on day 32 anddifferent samples were taken. 6 mice were either found dead or wereterminated early due to worsened overall condition.

4T1 lung metastases were determined by lung weight, lung tumor nodulequantification including nodule number, size and calculated tumorvolume, as well as histological quantification. Results are shown inFIG. 2. MONC 402 (20 mg/kg/day) monotherapy groups did not significantlyinhibit 4T1 lung metastasis. Cisplatin (1.25 mg/kg) monotherapy showedsignificant anti-tumor efficacy (p<0.05). The combination of Cisplatin(1.25 mg/kg) with MONC402 (20 mg/kg/day) displayed efficacy in reducinglung metastasis (p<0.0005) and reducing microvessel density.Importantly, the combination therapy group also showed better anti-tumorefficacy when compared to the cisplatin monotherapy group determined bylung weight (p<0.02), tumor nodule number, lung tumor coverage byhistology, and lung tumor microvessel density (p<0.05, t-test),demonstrating MONC402 enhanced the anti-tumor efficacy of cisplatin.

Model D: Human Prostate Carcinoma PC-3M Model: Combination Therapy

Male SCID/Beige mice 8 WOA were injected with 5×10⁵ PC-3M-luciferaseprostate carcinoma cells intra prostate. Daily treatment with saline orMONC402 with or without weekly treatment of cisplatin started on day 3.Mice were monitored weekly with Xenogen imaging system. The experimentwas terminated on day 32. Different organs were isolated and tumormetastasis was assessed by weight and Xenogen imaging.

The MONC402 (30 mg/kg) monotherapy inhibited PC-3M metastasis in theperitoneum. Cisplatin combined with MONC402 (30 mg/kg) decreased tumorgrowth compared to saline and MONC402 monotherapy groups as determinedby in vivo imaging. There was no significant difference betweencombination therapy (Cisplatin+MONC402 30 mg/kg) and Cisplatinmonotherapy in primary tumor weight and metastasis under the specificexperimental condition.

Example 4 More Combination Studies and Effect of MONC402 on Mobilizationof Cells from Bone Marrow

A. MONC402 Effect on Mobilization of Endothelial Progenitor Cells

Certain chemotherapeutic agents induce mobilization of endothelialprogenitor cells

(EPC). The therapeutic benefits of such agents may be compromised byinduction of EPC mobilization that promotes rapid tumor regrowth (see,e.g., Shaked et al., 2008 Cancer Cell, 14: 263-273). The effect ofMONC402 given in combination with agents that cause this phenomenon wasassessed.

In one experiment, normal mice were treated with G-CSF (s.c., 3consecutive days), docetaxel or cisplatin (one i.p. dose) with orwithout simultaneous MONC402 treatment (one s.c. dose). EPC mobilizationwas monitored 24 h later. Mice (8 mice/group) were dosed s.c. withSaline or MONC402 (40 mg/kg) (or DC101 at 40 mg/kg for control withdocetaxel). About 30 min later, mice were given docetaxel (i.p., 40mg/kg), cisplatin (i.p., 6 mg/kg), or saline control. As a positivecontrol, 2 groups of mice were also dosed with G-CSF i.p. (200 μg/kg)for 3 days, either alone or in combination with a single dose of MONC402(s.c. at 40 mg/kg) on the last day. 24 h later, the mice wereeuthanized, and 0.5-0.8 mL of blood taken by cardiac puncture:

About 300 μL of whole blood was transferred directly into 14 mL of lysisbuffer for flow cytometry of EPC. The remaining 300-600 μL wereprocessed for serum (SDF-1α). For flow cytometry, lysed cells werewashed and blocked, then stained with CD13-FITC, CD117-PE, 7-AAD,VEGFR2-APC, and CD45-PE/Cy7. A total of 50,000 cells were acquired persample in the PBMNC gate.

G-CSF and docetaxel induced a significant increase in EPC as compared tothe Saline control, while cisplatin treatment did not show this effect.As seen in FIG. 3, docetaxel-induced EPC mobilization was inhibitedsignificantly by DC101 (anti-VEGFR2Ab) and MONC402. MONC402 did notinfluence EPC mobilization in saline or cisplatin treated mice.

The effect of MONC402 on EPC mobilization with G-CSF was smaller. Thisillustrates synergistic effects of MONC402 in combination with a taxaneand with GCSF.

In a second experiment, it was tested if daily dosing for 5 days withMONC402 would generate a stronger inhibitory effect on EPC mobilizationcaused by treatment with a taxane.

Mice (10 mice/group) were dosed s.c. with Saline or MONC402 (40 mg/kg),or DC101 (40 mg/kg, as a positive inhibition control with docetaxel).About 30 min later, mice were given docetaxel (i.p., 40 mg/kg) or salinecontrol. As a positive control, 2 groups of mice were also dosed withG-CSF (200 μg/kg) s.c. for 5 consecutive days±daily MONC402. 24 h later,the mice were euthanized, and 500-800 μL of blood taken by cardiacpuncture. 150 μL were transferred into 5 mL of lysis buffer for flowcytometric analysis of EPC. The remaining 350-500 μL were processed forserum (SDF-1α). For flow cytometry, cells were washed and blocked andthen stained with CD13-FITC, CD117-PE, 7-AAD, VEGFR2-APC, andCD45-PE/Cy7. A total of 50,000 cells were acquired per sample in thePBMNC gate.

As shown in FIG. 4, G-CSF and Docetaxel induced a significant increasein EPC mobilization, and both could be inhibited by MONC402. MONC402 wasequivalent to DC101 (anti-VEGFR2Ab) in inhibiting the docetaxel inducedEPC mobilization. MONC402 did not influence EPC mobilization in salinetreated mice, indicating that MONC402 does not suppress normal EPCgeneration or release, but may interfere with mechanisms induced bydocetaxel. Again, this shows MONC402's synergistic effects incombination with agents that induce EPC mobilization.

A third experiment was conducted to evaluate the effect of MONC402 onEPC mobilization in response to treatment with docetaxel in 4T1 tumorbearing mice. The study also evaluated if EPC mobilization could beinhibited by MONC402 administered via osmotic pump.

4T1 tumors were implanted on Day 0 at 1×10⁵ cells/mouse into the 4^(th)mammary fat pad. Pumps with Saline or MONC402 were implanted on Day 0,immediately after tumor cell injections. Mice were randomized to thefollowing groups:

1. Saline Control, n=8

2. Docetaxel, 40 mg/kg, Day 6, n=8

3. M402, pump, 40 mg/kg/day, Day 0, n=8

4. Docetaxel, 40 mg/kg, Day 6+M402, pump, 40 mg/kg/day, Day 0, n=8

Docetaxel or Saline was administered i.p. on Day 6 in the AM. Four hlater, mice were bled by submandibular plexus for EPC profiling andsoluble factor analysis. Primary tumors were removed on Day 9 forhistological analyses. Blood was also collected for soluble factoranalyses (72 h after docetaxel dosing). Serum samples were analyzedusing a 19-plex Luminex kit. In addition, samples were also tested forSDF-1α levels by ELISA. SDF-1α was chosen because it is aheparin-binding protein; publications showed an increase in thischemokine upon docetaxel treatment and SDF-1α is involved in recruitingbone marrow stem cells to new sites. MONC402 and the combination ofMONC402 with Docetaxel had a small, non-significant impact on tumorweight.

Docetaxel induced a significant increase in EPC in the blood by 4 h atthe 40 mg/kg dose, as observed in previous studies. MONC402 monotherapy,delivered via osmotic pump at 40 mg/kg/day did not induce significantchanges in mobilized EPC; however, a trend toward reduced EPC levels wasobserved. As shown in FIG. 5, the co-administration of MONC402 bycontinuous osmotic pump significantly inhibited the blood EPCmobilization by docetaxel and reduced the levels to those of the salinecontrol in 7 of 8 mice. These results confirm the observations from theprevious studies, this time conducted in tumor-bearing mice and byadministering MONC402 via osmotic pump. Taken together, these datasuggest that a single docetaxel dose mobilizes EPC into the blood, whichcan be inhibited with concurrent MONC402 treatment at 40 mg/kg/day.

B. MONC402 Effect on Mobilization of G-CSF Induced MDSC Mobilization

The effect of MONC402 on mobilization of other cells from thebone-marrow was assessed.

BALB/c mice were treated with 4 daily doses of sc saline or G-CSF at 0.5mg/kg, in combination with sc MONC402 (20 mg/kg). Animals weresacrificed on day 5, blood samples saved by cardiac puncture. Hematologyanalyses were performed with VetScan HM2. Granulocytes (GRA) are definedby size and granularity, and >90% were CD11b⁺GR-1⁺ when analyzed by flowcytometry. As shown in FIG. 6, MONC402 inhibited G-CSF inducedmobilization of MDSC.

C. Effect of Combination with Tyrosine Kinase Inhibitors

This example tested the effect of MONC402 in combination with a tyrosinekinase inhibitor used in treatment of cancer: sunitinib (Sutent®) at twodifferent time points. A dose of sunitinib at 90 mg/kg was chosen as iteffectively mobilized EPC without causing toxicity in this mouse model.

Accelerated tumor invasion and metastasis after short term sunitinibtreatment is reported in the literature (Ebos et al. 2009. Cancer Cell15: 232-239). Surprisingly, as described below, the addition of MONC402to the sunitinib treatment significantly inhibited EPC mobilization andreduced the aggressive regrowth phenotype in tumor-bearing mice. Whilenot bound by theory, analysis of EPC in the bone marrow suggests thatMONC402 may prevent the egress of bone marrow progenitor cells intocirculation.

In one experiment, 36 Balb/c female mice were treated daily with vehicleor 90 mg/kg sunitinib (8 mice per group). Sunitinib was administeredorally as a suspension in vehicle (0.5% carboxy methyl cellulose, 0.4%Tween 80, 1.8% NaCl). MONC402 (20 mg/kg for a 20 g mouse) wasadministered s.c. twice daily.

Group # 1 N = 8 Vehicle (0.1 ml p.o. opd) Saline (0.2 ml s.c. bid) Group# 2 N = 8 Sunitinib 90 mg/kg Saline (0.2 ml s.c. bid) (0.1 ml p.o. opd)Group # 3 N = 8 Vehicle (0.1 ml p.o. opd) MONC402 20 mg/kg (0.2 ml s.c.bid) Group # 4 N = 8 Sunitinib 90 mg/kg MONC402 20 mg/kg (0.1 ml p.o.opd) (0.2 ml s.c. bid)

On day 7 four mice in each group were sacrificed 2 hours after the lastdose of sunitinib and MONC402. Whole blood was collected for EPCisolation as well as sera and bone marrow from one femur for each mouse.Isolation and FACS analysis of EPCs was done. Sera was separated andstored at −80° C. until Luminex analysis or SDF-1α or SCF ELISA.

On day 8 four mice in each group were sacrificed 24 hours after the lastdose of sunitinib and MONC402. Whole blood was collected for EPCisolation as well as sera and bone marrow from one femur for each mouse.Isolation and FACS analysis of EPCs was done. Sera was separated andstored at −80° C. until Luminex analysis or SDF-1α or Stem Cell Factor(SCF) ELISA.

Sunitinib induced a significant increase in EPC in the blood by 2 h.This increase was transient and resolved by 24 h in this study. MONC402monotherapy at 20 mg/kg BID did not induce significant changes at 2 h or24 h in blood EPC. At 2 h, the addition of MONC402 to the sunitinibtreatment significantly inhibited the blood EPC mobilization and reducedthe levels to those of the saline control in 2 of 4 mice. Both sunitiniband sunitinib in combination with MONC402 induced a significant increasein the production of EPC in the bone marrow at 2 h, which wassignificantly higher in the mice receiving sunitinib and MONC402. By 24h, the percentage of EPC in the bone marrow was normalized in the groupreceiving sunitinib and close to the saline control in the groupreceiving sunitinib and MONC402. No increase in the production of EPC inthe bone marrow was observed at 2 h or 24 h for mice treated withMONC402 alone.

Taken together, the data suggest that sunitinib treatment for 7 daysmobilizes EPC into the blood, which can be inhibited with concurrentMONC402 treatment at 20 mg/kg BID. While not bound by theory, MONC402may act via trapping the induced EPC in the bone marrow.

A second experiment was performed to demonstrate the effect of MONC402on sunitinib (Sutent®) induced EPC mobilization in tumor-bearing mice.

Luciferase-transfected MB-231-3P cells were implanted into 20 cages of8-9 week old female Fox-Chase SCID mice at a concentration of 7×10⁵cells/60 μL/mouse on Day 0. Primary tumor volume was monitored throughDay 21. Tumors were resected and weighed on Day 25. Mice were randomizedinto 4 groups:

-   -   Vehicle control    -   Sunitinib (60 mg/kg once a day for 7 days, po)    -   MONC402 (20 mg/kg twice a day for 7 days, sc)    -   Combo therapy (sunitinib and MONC402)

Animals in which primary tumors had attached to or penetrated theabdominal muscle or where the abdominal muscle was compromised duringsurgery were not used in this experiment. Treatment commenced on Day 26,one day after primary tumor resection. Each morning, sunitinib orvehicle was administered to all animals via oral gavage. MONC402 orsaline was then administered via subcutaneous injection. Animals wereagain administered MONC402 or saline later in the afternoon. Sunitinibwas prepared fresh daily by suspending the contents of one (1) 50 mgcapsule in 8.3 mL vehicle. Sunitinib was resuspended by vortex beforegavaging each animal. Wet food was provided daily to all animals. Tumorswere resected on Day 25 and treatment began on Day 26.

Mice were bled via submandibular plexus 24 h after the last sunitinibdose and one drop of blood collected into RBC lysis buffer. Cells werewashed twice with FACS buffer and stained with: anti-CD13-FITC,anti-CD117-PE, 7-AAD, anti-VEGFR-2-APC and anti-CD45-PE/Cy7 for 20 minat 2-8° C. Cells were washed again and fixed in 2% formaldehyde. Sampleswere analyzed on the FACS Canto the following day.

Sunitinib monotherapy showed a modest but significant increase incirculating EPC 24 h after dosing. The increase was not as pronounced asobserved with the higher (120 mg/kg) Sunitinib dose in normal mice andwas mainly driven by 4 mice with higher percentages. MONC402 treatmentdid not increase the percentage of circulating EPC as compared to thesaline control. The combination of MONC402 with sunitinib resulted incirculating EPC levels similar to the sunitinib alone group, againdriven by 4 mice with higher percentages. See FIG. 7.

In a third experiment, Luciferase-transfected MB-231-3P cells wereimplanted into 21 cages of 10-11 week old female Fox-Chase SCID mice ata concentration of 1×10⁶ cells/50 μL/mouse on Day 0. The effect ofMONC402 on Sutent-induced accelerated tumor regrowth and metastasis wasevaluated. Primary tumor volume was monitored through Day 21. Tumorswere resected on Day 24 after implant at which time tumors were316.9±11.1 mm³. Mice with lower body weight (≦17-18 g) were excludedfrom the study. Animals were subsequently randomized into 1 of 4 groups:(1) Vehicle control; (2) Sutent (60 mg/kg QDx7 po); (3) M402 (20 mg/kgBIDx7 sc); (4) Combo therapy (Sutent QD+M402 BID). Treatments commencedon Day 26, two days after primary tumor resection, and were given for 7consecutive days. Mice received no further treatment thereafter and weremonitored for tumor progression by bioluminescent imaging and survival.FIG. 8 depicts whole body bioluminescence over time (Mean±SEM, n=16).Sutent treated animal displayed significantly accelerated tumorprogression (primary tumor re-growth and metastasis) when compared tosaline control group (P<0.05, One-way ANOVA with Newman-Keuls multiplecomparison test). M402 treated animals did not show significant changesin tumor progression when compared to saline control. Most importantly,when M402 was given together with Sutent, it significantly delayed tumorprogression when compared to Sutent monotherapy group (P<0.05, One-wayANOVA with Newman-Keuls multiple comparison test).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-36. (canceled)
 37. A method of lessening, reducing, mitigating,ameliorating, or delaying pancreatic cancer in a subject, comprisingadministering to the subject a pharmaceutical composition comprising apolysaccharide preparation having the following characteristics: (a) aweight average chain molecular weight between 3,500 and 8,000 Da; (b)anti-Xa activity of less than 20 IU/mg and anti-IIa activity of 1 IU/mgor less; (c) greater than 5% and less than 25% glycol split uronic acidresidues; and wherein, the preparation comprises polysaccharides thatcomprise the structure:[U _(w) −H _(x,y,z)]_(m) ˜[U _(G) −H _(x,y,z)]_(n), or apharmaceutically acceptable salt thereof, wherein each occurrence of Uindicates a uronic acid residue and each occurrence of H indicates ahexosamine residue; wherein m and n are integers such that m=4-16, andn=1-4; each of w, x, y and z can independently be the same or differentfor each occurrence of [U_(w)−H_(x,y,z)] and each of x, y and z canindependently be the same or different for each occurrence of[U_(G)−H_(x,y,z)], wherein w=−2OS or −2OH; x=—NS or —NAc; y=−3OS or−3OH; z=−6OS or −6OH; and

wherein the symbol ˜ indicates that the units marked m and n aredistributed along the polysaccharide chain and are not necessarily insequence; and wherein the polysaccharide preparation is administered incombination with abraxane and gemcitabine.
 38. The method of claim 37,wherein the pancreatic cancer is a metastatic cancer.
 39. The method ofclaim 37, wherein the pancreatic cancer is a locally advanced prostatecancer.
 40. The method of claim 37, wherein the preparation isadministered intravenously.
 41. The method of claim 37, wherein thepreparation is administered subcutaneously.
 42. The method of claim 37,wherein the preparation is administered daily.
 43. The method of claim37, wherein the subject receives multiple administrations of thepreparation and the preparation is administered daily.
 44. The method ofclaim 37, wherein the preparation, gemcitabine, and abraxane areadministered simultaneously.
 45. The method of claim 37, wherein theweight average chain molecular weight of the preparation is between4,000 and 8,000 Da.
 46. The method of claim 37, wherein the weightaverage chain molecular weight of the preparation is between 5,000 and8,000 Da.
 47. The method of claim 37, wherein the preparation hasgreater than 5% and less than 20% glycol split uronic acid residues. 48.The method of claim 37, wherein the preparation has between 10% and 20%glycol split uronic acid residues.
 49. The method of claim 37, whereinthe preparation has greater than 40% U_(2S)H_(NS,6S) disaccharideresidues.
 50. The method of claim 49, wherein the preparation hasgreater than 70% U_(2S)H_(NS,6S) disaccharide residues.
 51. The methodof claim 49, wherein the preparation has a degree of desulfation lessthan 40%.
 52. The method of claim 49, wherein the preparation has adegree of desulfation less than 30%.
 53. The method of claim 49, whereinthe preparation has a degree of desulfation less than 10%.
 54. Themethod of claim 37, wherein the polysaccharide preparation has ananti-Xa activity of less than 15 IU/mg.
 55. The method of claim 37,wherein the polysaccharide preparation has an anti-Xa activity of lessthan 10 IU/mg.
 56. The method of claim 37, wherein the reducing end ofthe polysaccharides comprise a 2,5-anhydromannitol residue.
 57. Themethod of claim 37, wherein about 50% of the reducing ends of thepolysaccharides comprise a 2,5-anhydromannitol residue.
 58. The methodof claim 37, wherein the polysaccharides of the preparation have auronic acid at the non-reducing end.
 59. The method of claim 58, whereinthe polysaccharides of the preparation have a non-native uronic acid atthe non-reducing end.
 60. The method of claim 58, wherein thepolysaccharides of the preparation have a glycol split uronic acid atthe non-reducing end.
 61. The method of claim 37, wherein n=1-3.
 62. Amethod of lessening, reducing, mitigating, ameliorating, or delayingpancreatic cancer in a subject, the method comprising subcutaneousadministration of a polysaccharide preparation having (a) anti-Xaactivity of less than 20 IU/mg and anti-IIa activity of 1 IU/mg or less;(b) greater than 5% and less than 25% glycol split uronic acid residues;and wherein, the preparation comprises polysaccharides that comprise thestructure: [U_(w)−H_(x,y,z)]_(m)˜[U_(G)−H_(x,y,z)]_(n), or apharmaceutically acceptable salt thereof, wherein each occurrence of Uindicates a uronic acid residue and each occurrence of H indicates ahexosamine residue; wherein m and n are integers such that m=4-16, andn=1-4; each of w, x, y and z can independently be the same or differentfor each occurrence of [U_(w)−H_(x,y,z)] and each of x, y and z canindependently be the same or different for each occurrence of[U_(G)−H_(x,y,z)], wherein w=−2OS or −2OH; x=—NS or —NAc; y=−3OS or−3OH; z=−6OS or −6OH; and

wherein the symbol ˜ indicates that the units marked m and n aredistributed along the polysaccharide chain and are not necessarily insequence; and wherein the polysaccharide preparation is administered incombination with abraxane and gemcitabine.
 63. The method of claim 62,wherein the pancreatic cancer is a locally advanced metastatic cancer.64. The method of claim 62, wherein the subject receives multipleadministrations of the preparation and the preparation is administereddaily.
 65. The method of claim 62, wherein the preparation has amolecular weight distribution such that 10-40% of the polysaccharides ofthe preparation have a molecular weight<3000 Da; 45-65% of thepolysaccharides have a molecular weight between 3000-8000 Da, and 15-30%of the polysaccharides have a molecular weight>8000 Da.
 66. The methodof claim 62, wherein the plurality of polysaccharide chains of thepreparation each have no more than 2 glycol split uronic acid residues(U_(G)).
 67. The method of claim 66, wherein the plurality ofpolysaccharide chains of the preparation each have no more than 1 glycolsplit uronic acid residue (U_(G)) per polysaccharide chain.
 68. Themethod of claim 62, wherein the polysaccharide preparation has ananti-Xa activity of less than 15 IU/mg.
 69. The method of claim 62,wherein the polysaccharide preparation has an anti-Xa activity of lessthan 10 IU/mg.
 70. The method of claim 62, wherein the reducing end ofthe polysaccharides comprise a 2,5-anhydromannitol residue.
 71. Themethod of claim 62, wherein about 50% of the reducing ends of thepolysaccharides comprise a 2,5-anhydromannitol residue.
 72. The methodof claim 62, wherein the polysaccharides of the preparation have auronic acid at the non-reducing end.
 73. The method of claim 72, whereinthe polysaccharides of the preparation have a non-native uronic acid atthe non-reducing end.
 74. The method of claim 72, wherein thepolysaccharides of the preparation have a glycol split uronic acid atthe non-reducing end.
 75. The method of claim 62, wherein m=4-14. 76.The method of claim 62, wherein m=4-13.
 77. The method of claim 62,wherein n=1-3.
 78. The method of claim 62, wherein n=1-2.
 79. A methodof lessening, reducing, mitigating, ameliorating, or delaying locallyadvanced or metastatic pancreatic cancer in a subject, comprisingadministering to the subject a pharmaceutical composition comprising apolysaccharide preparation having the following characteristics: (a) aweight average chain molecular weight between 3,500 and 8,000 Da; (b)anti-Xa activity of less than 20 IU/mg and anti-IIa activity of 1 IU/mgor less; (c) greater than 5% and less than 25% glycol split uronic acidresidues; and wherein the preparation comprises polysaccharides thatcomprise the structure: [U_(w)−H_(x,y,z)]_(m)˜[U_(G)−H_(x,y,z)]_(n), ora pharmaceutically acceptable salt thereof, wherein each occurrence of Uindicates a uronic acid residue and each occurrence of H indicates ahexosamine residue; wherein m and n are integers such that m=4-15, andn=1-3; each of w, x, y and z can, independently, be the same ordifferent for each occurrence of [U_(w)−H_(x,y,z)] and each of x, y andz can, independently, be the same or different for each occurrence of[U_(G)−H_(x,y,z)], wherein w=−2OS or −2OH; x=—NS or —NAc; y=−3OS or−3OH; z=−6OS or −6OH; and

wherein the symbol ˜ indicates that the units marked m and n aredistributed along the polysaccharide chain and are not necessarily insequence; and wherein a plurality of the polysaccharides in thepreparation have a 2,5-anhydromannitol residue at the reducing end and anon-native uronic acid at the non-reducing end; and wherein the subjectreceives multiple administrations of the polysaccharide preparation, thepolysaccharide preparation is administered daily for at least one month,and the polysaccharide preparation is administered in combination withabraxane and gemcitabine.
 80. The method of claim 79, wherein thepreparation is administered subcutaneously.